Within the frame of the de novo formation of Platelet-Activating Factor in Tetrahymena, the occurrence as well as the properties of a lipid phosphate phosphohydrolase activity catalyzing the dephosphorylation of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate was investigated. The activity was distributed in all the membrane fractions of the cell and in the cytosol. It showed preference for acyl-acetyl-sn-glycero-phosphate as well, and at a much lower level, for dipalmitoyl-glycero-phosphate. Mg² and Ca² caused a dose-dependent inhibition, while F⁻, EDTA and EGTA had no effect. The enzymic activity was linear for at least up to 60 min incubation time and up to 150 μg protein. Microsomal activity exhibited two optimal pH areas, around 7.0 and 9.0, while mitochondrial activity showed one peak, at pH 7.0. Acyl-GP, acyl-acetyl-GP and alkyl-GP could replace alkyl-acetyl-GP in significant rates, while dipalmitoyl-GP, β-GP, fructose-6-GP, p-nitrophenylphosphate, creatine phosphate or ATP had no effect. Side phospholipase A² and C activities were also detected. Taking into account the presence of PAF and alkylacetylglycerol in the protozoan as well as the presence of a dithiothreitol-insensitive CDP-choline:cholinephosphotransferase activity that converts alkylacetylglycerol to PAF, we suggest that the present phosphohydrolase activity may be involved in the de novo production of PAF within Tetrahymena.