Glycosyl-phosphatidylinositol (GPI)-anchored plasma membrane proteins have been found to be widespread in eukaryotes and protozoa but have not been reported in higher terrestrial plants. A sensitive biotin-based assay has been used to detect the presence of GPI-anchored proteins on the outer surface of cultured Nicotiana tabacum cells. Six proteins with molecular weights of 92, 84, 60.5, 54.5, 39.5 and 37 kDa were found to move from a Triton X-114 detergent-rich phase to an aqueous phase following incubation with phosphatidylinositol-specific phospholipase C (PtdIns-PLC). The behaviour of these proteins is consistent with the presence of a GPI-anchor. Seven GPI-anchored proteins were also detected on the surface of tobacco leaf protoplasts with molecular weights of 67.5, 62, 39, 33.5, 27, 23 and 15.6 kDa. These data demonstrate the presence of multiple GPI-anchored proteins on the plasma membrane of higher plant cells.