The ciliated protozoan Tetrahymena pyriformis contains platelet-activating factor (PAF) as a physiological minor lipid. Its subcellular localization was found as follows: 13.7% in the pellicles, 24.9% in mitochondria, 56.5% in microsomes and 7.1% in the cytosol. Succinate dehydrogenase was used as marker enzyme. PAF remains cell-associated unless bovine serum albumin is included in the extracellular medium. In this case 15% of total PAF, portion comparable to that found in the pellicles, is released. Investigation of the principal enzymic activities involved in PAF formation showed that PAF-acetyltransferase (2.3.167) is totally absent from the protozoan. This means that the 'remodelling' pathway occurring in pro-inflammatory cells does not contribute in PAF formation in our system. A dithiothreitol (DTT)-insensitive CDPcholine phosphocholinetransferase activity involved in PAF biosynthesis is shown for the first time to be responsible for PAF production in T. pyriformis. It uses exogenous alkyl-acetyl-glycerol as substrate and is saturated over substrate concentration 250 microM. It can also use endogenous lipids as substrate. It is distributed mainly in mitochondria and microsomes, much less is found in the pellicles and it is totally absent from the cytosol. Its insensitivity to DTT, its selectivity to alkyl-acetyl-G and its different distribution compared to the enzymic activity involved in PC formation (EC 2.7.8.2) suggest that a different enzyme, specific for PAF formation (EC2.7.8.16) via the de novo pathway exists in the protozoan.