The addition of platelet-activating factor to horse platelets prelabeled with (32P)orthophosphate induces the rapid formation of [32P]phosphatidic acid. This is subsequently followed by a considerable increase in the labeling of phosphatidylinositol. Activation of the formation of phosphatidic acid by platelet-activating factor is evident at a concentration of 1 nM, is maximal at 0.1 microM, and independent of the presence or absence of plasma in the medium. In horse platelets prelabeled with [14C]arachidonic acid, platelet-activating factor stimulates the rapid formation of [14C]phosphatidic acid, [14C]arachidonic acid, and [14C]arachidonate metabolites. Concomitantly, there is a loss of radioactivity from phosphatidylinostiol., phosphatidylcholine, and phosphatidylethanolamine. Platelet-activating factor at a concentration of 1 nM stimulates formation of phosphatidic acid while the appearance of arachidonate metabolites is seen at a higher concentration (10 nM), without acetate in the 2-position, is unable to induce in platelets the formation of phosphatidic acid, arachidonate metabolites, or the release of [3H]serotonin and, in addition, does not antagonize the action of platelet-activating factor. the release of [3H]serotonin and [14C]arachidonate from platelets stimulated with platelet-activating factor is not affected by indomethacin. Trifluoperazine (50-100 microM) inhibits the platelet-activating factor-stimulated liberation of arachidonic acid from phospholipids, without affecting the formation of phosphatidic acid and the release of serotonin. Prostacyclin, on the other hand, inhibits the platelet-activating factor-induced release of serotonin, arachidonate metabolites, and formation of phosphatidic acid. These data indicate a close relationship between the formation of phosphatidic acid and the release of serotonin in platelets stimulated with the platelet-activating factor.