Cultured human endothelial cells produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) when stimulated with human thrombin. The response to thrombin is dose dependent, with a half-maximal effect at 0.17 unit/ml. The product is identified as PAF by the incorporation of radiolabeled precursors, its behavior in chromatographic systems, the recovery of biological activity, and the effect of treatment with phospholipase A2. Incorporation of [3H]acetate into PAF is maximal by 5 min and decreases thereafter. Endothelial cells produce 0.10-0.17 nmol of PAF per 10(6) cells in a 5-min exposure to thrombin, as judged by the amount of neutrophil-aggregating activity. The production of this potent agonist for platelet activation and neutrophil chemotaxis by endothelial cells suggests that it may play a role in the maintenance of vascular integrity and perhaps in pathological events such as thrombosis and atherogenesis.