Particulate preparations of human platelets were capable of catalyzing acylation of sn-glycerol 3-phosphate by long-chain fatty acyl-CoA thioesters. The principal lipid product formed was identified as phosphatidic acid. The highest specific activity was found in the particulate fraction that sedimented between 12,000 g and 105,000 g. The reaction exhibited a broad pH optimum around 7.4-8.5. The apparent Michaelis constant for sn-glycerol 3-phosphate was 0.48 mM when 28.5 µM palmityl-CoA was used as acyl donor. Palmityl- and oleyl-CoA were better substrates than stearyl-, linoleyl-, and arachidonyl-CoA, as far as the maximal velocity was concerned. Particulate preparations of platelets from normal subjects catalyzed the incorporation of 0.271 ± 0.048 nmole of sn-glycerol 3-phosphate/min per mg of protein. The capacity of human platelets to acylate sn-glycerol 3-phosphate was approximately 40% that of human liver, as compared on the basis of the specific activity of the microsomal fraction. These results suggest that the glycerophosphate pathway makes an essential contribution to the de novo synthesis of phospholipids in human platelets.