In this study, the molecular species distribution of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) synthesized by rabbit peritoneal and human peripheral neutrophils was examined. Radiolabeled platelet-activating factor synthesized from [3H]acetate by neutrophils stimulated with ionophore A23187, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was separated into the individual molecular species on the basis of length and degree of unsaturation of the 1-O-alkyl chain using reverse-phase high-performance liquid chromatography. The predominant alkyl chains in the labeled platelet-activating factor synthesized by ionophore- or zymosan-stimulated rabbit cells were 15:0 (4%), 16:0 (43%), 18:0 (11%), and 18:1 (26%). This is in contrast to the alkyl chain distribution of the widely accepted precursor of platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, present in the cell. The major alkyl chains contained in the labeled platelet-activating factor synthesized by ionophore-, zymosan-, or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils were 16:0 (40%), 17:0 (8 and 5%; two isomers), 18:0 (16%), and 18:1 (18%). As found with the rabbit cells, this distribution differs from the alkyl chain content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine in human neutrophils, which contains less than 1% of each 17:0 isomer. We demonstrate here that the platelet-activating factor synthesized by rabbit peritoneal and human peripheral polymorphonuclear neutrophils is heterogeneous and that some selectivity exists in the choice of the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine precursor for platelet-activating factor synthesis in these cells. It also appears that the molecular species distribution of platelet-activating factor is independent of the stimulus used to elicit its synthesis.