In order to detect novel glycophosphatidylinositol (GPI)-anchored platelet proteins, human platelets were incubated with PI-specific phospholipase C (PI-PLC) and the supernatant was analysed by PAGE and silver-staining for additional protein bands. PI-PLC treatment resulted in the appearance of at least two additional novel GPI-linked glycoproteins (GP), GP500 and GP175, in the supernatant. Their presence on the platelet plasma membrane surface was demonstrated by periodate/[3H]borohydride surface-labelling. Activation of platelets did not enhance the amount of GP500 and GP175 that could be cleaved by PI-PLC. In Triton X-114 phase partitioning of platelet membranes the membrane form of GP175, mfGP175, was in the Triton phase while mfGP500 was found in the water phase. Neither GP500 nor GP175 were present in the supernatant of surface-labelled platelets treated with PI-PLC from 4 patients, diagnosed as having paroxysmal nocturnal haemoglobinuria (PNH), but the supernatant from platelets from healthy volunteers treated the same way contained both.