The lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was biosynthesized, purified and incubated with washed human platelet. It inhibited arachidonic acid, azo-prostaglandin H2 or U-46619-induced aggregation and secretion in a concentration-dependent fashion (IC50 = 2-3 microM). Collagen-induced aggregation and secretion were also inhibited (IC50 = 6 microM). 12-HPETE inhibited malondialdehyde and thromboxane B2 formation in platelets stimulated with arachidonic acid or thrombin. While thrombin-induced aggregation was unaffected by 50 microM 12-HPETE, thrombin-induced secretion was inhibited. Inhibiton of secretion by 12-HPETE was observed in platelets from untreated as well as aspirin-treated donors, indicating that 12-HPETE inhibits secretion independently of its ability to inhibit prostaglandin formation. Aggregation of washed human platelets by arachidonic acid yielded a bell-shaped concentration-response curve. Diminished aggregation at higher concentrations was associated with an increase in the ratio of lipoxygenase products to thromboxane B2. The data suggest that 12-HPETE formation may regulate platelet aggregation and secretion and that its primary effect, at low concentrations, is inhibition of endoperoxide-induced responses. At higher concentrations 12-HPETE also inhibits arachidonic acid metabolism. Thus, the combined inhibitory effects on endoperoxide-induced aggregation and thromboxane formation would explain the diminished aggregation observed in response to high concentrations of arachidonic acid.