We have analyzed the effects of exogenous phospholipase C (PLC) on the cell-surface polypeptides and proteinases of Herpetomonas samuelpessoai grown in chemically defined conditions by SDS-PAGE gels. Live parasites treated with PLC released into the extracellular medium a complex profile of glycosylphosphatidylinositol (GPI)-anchored polypeptides ranging from 15 to 100 kDa, some of them with proteolytic activity. Two major metalloproteinases with apparent molecular masses of 63 and 115 kDa were observed after PLC hydrolysis. Interestingly, only the PLC-released soluble form of the 115-kDa metalloenzyme, and not the me membrane-anchored form, displayed proteolytic activity, demonstrating that cleavage of the GPI anchor can lead to enzymatic activation.