Arachidonate and other unsaturated fatty acids stimulated platelet protein kinase C in a dose-dependent manner (5-50 micrograms/ml), when the activity was assayed with either isolated substrates or the platelet cytosol. When human platelets were stimulated by arachidonate, two types of platelet activation were observed. Platelet activation induced by a low level of arachidonate (0.1-5 micrograms/ml) was inhibited by aspirin, but activation induced by a high level of arachidonate (10-50 micrograms/ml) was not. These activations were associated with the phosphorylation of 40K and 20K proteins. Other unsaturated fatty acids (10-50 micrograms/ml) also induced platelet aggregation which was not inhibited by aspirin. Arachidic acid and methyl arachidonate, which did not stimulate protein kinase C, also did not induce platelet responses. Although a low level of arachidonate (0.45 microgram/ml) induced the rapid and transient formation of [3H]-1,2-diacylglycerol and [32P]phosphatidate in intact platelets with [3H]arachidonate or [32P]Pi, unsaturated fatty acids at a high concentration (50 micrograms/ml) did not stimulate phospholipase C. Incubation of fura 2 loaded platelets with a high level of unsaturated fatty acids evoked a rise in cytosolic Ca2+-concentration ([Ca2+]i) but this [Ca2+]i elevation alone was not associated with platelet activation. These results suggest that a high level of unsaturated fatty acids induces platelet activation, without phospholipase C stimulation, and that the ability of unsaturated fatty acid to directly activate protein kinase C may contribute toward the activation of platelets by a high level of unsaturated fatty acid.