It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.