Summary. For many years endocytosis has been regarded with great scepsis by plant physiologists. Although now generally accepted, care must still be taken with experiments designed to demonstrate endocytic uptake at the plasma membrane. We have taken a critical look at the vari- ous agents which are in use as markers for plant endocytosis, pointing out pitfalls and precautions which should be taken. We also take this op- portunity to introduce the tyrphostins – tyrosine kinase inhibitors –, which also seem to prevent endocytosis in plants. Keywords: Biotinylated marker; FM lipophilic styryl dye; Lucifer Yellow; Tyrphostin; Wortmannin. Introduction In 1980 W. J. Cram made the unfortunate statement, “We shall argue firstly, on ultrastructural and physiological grounds, that pinocytosis does not occur and secondly, on theoretical grounds, that it could not occur in a turgid plant cell’’ (Cram 1980: p. 1). 25 years on and a good dozen reviews later it can safely be said that endocytosis is not restricted to mammalian cells but is also exhibited by walled organisms, including plants. Nevertheless, it has to be admitted that as far as plants are concerned, the unequivocal demonstration of the internalization of a re- ceptor-ligand complex through a clathrin-coated pit and the recycling of the receptor to the plasma membrane (PM) still remains to be published. Despite the fact that, from the structural view point, it has been known for al- most 20 years that plant cells possess endocytic organelles (clathrin-coated pits and vesicles, multivesicular bodies), the commonly held belief among physiologists that turgor pressure in plants would make endocytosis difficult, if not impossible, has delayed the appreciation of this process by the plant community. The lack of obvious molecules for endocytic cargo has not helped this situation. Turgor no longer plays an impeding role for endocytosis in plants (Saxton and Breidenbach 1988, Meckel et al. 2004), and a number of good candidates for internalizable receptors at the PM are now known (Holstein 2002, Geldner 2004). One of the major problems in studies on endocytosis in plants arises from trying to determine whether a molecule has entered cells by a fluid-phase or a receptor-mediated pathway. Another problem lies in interpreting the results of the application of inhibitors, whose specificities may be wide and whose targets may differ throughout the endocytic path- way. A widely used indicator to discriminate between fluid- phase endocytosis (FPE) and receptor-mediated endocytosis (RME) is the kinetics of uptake. FPE shows linear uptake ki- netics with respect to increasing substrate concentration (an inevitable consequence of the nonselective uptake of external fluid) and shows no evidence of saturation over a large exter- nal solute range. In contrast, RME saturates with ligand con- centration (a consequence of the limited numbers of receptor molecules at the PM). Uptake of labelled solute by FPE is not reduced in the presence of unlabeled solute, while unla- belled free ligand competes for the uptake of labelled ligand during RME. Finally, the presence of a PM receptor allows ligand binding to the PM at either 4 °C or 37 °C, while bind- ing to the cell surface of a solute internalized by FPE is not detectable at either 4 °C or 37 °C. In this article we present a critical overview of the agents which have been used to demonstrate the process of endo- cytosis in plants, including markers and inhibitors. Protoplasma (2005) 226: 3–11 DOI 10.1007/s00709-005-0101-y PROTOPLASMA Printed in Austria Testing for endocytosis in plants F. Aniento 1 and D. G. Robinson 2, * 1 Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Valencia,Valencia 2 Heidelberg Institute for Plant Sciences, University of Heidelberg, Heidelberg Received February 7, 2005; accepted March 30, 2005; published online October 20, 2005 © Springer-Verlag 2005 * Correspondence and reprints: Heidelberg Institute for Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Federal Republic of Germany.