We added arachidonic acid (AA) and eicosapentaenoic acid (EPA) to washed platelet suspensions in the absence of albumin, holding the total amount of the fatty acids constant at 2 microM, and changing the ratio of EPA to AA. Platelet aggregation, serotonin release and the amount of thromboxane (TX) B2, a cyclooxygenase product synthesized from exogenous AA, decreased as the ratio was increased. The decreases were greater than the expected ones from the diminution of the amount of exogenous AA. On the other hand, 12-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product synthesized from exogenous AA, increased in the presence of EPA. Although EPA was reported to be a poor substrate for platelet cyclooxygenase, the amount of TXB3 synthesized from exogenous EPA increased markedly by the simultaneous addition of AA. These results suggest that the EPA/AA ratio-dependent decrease in platelet aggregation and serotonin release is caused at least by both the decrease in the absolute amount of AA and the inhibitory effect of EPA on AA-metabolism via the cyclooxygenase pathway. Further studies on effects of EPA-metabolites via the cyclooxygenase pathway on platelet responses will be needed.