The effect of hydroperoxy and hydroxy derivatives of various fatty acids on human platelet aggregation was determined to delineate potencies and structure-activity function. In this regard, the 22-carbon n-3 fatty acids are the most potent inhibitors in comparison to the n-6 lipoxygenase derivatives. Submicromolar levels of the docosapentaenoic (22:5) and especially docosahexaenoic (22:6) n-3 hydroperoxy and hydroxy derivatives specifically antagonize the platelet aggregating effect to arachidonic acid (AA, 20:4n-6) but not that of ADP or collagen. Chain length (22-C > 20-C), double-bond position (n-3 > n-6), and double-bond number (6 > 5 > 4) influence the degree of inhibition of AA-induced aggregation of human platelets. Moreover, significant differences in potency were associated with specific structural aspects of 22:6n-3 lipoxygenase derivatives of 22:6n-3 as follows: functional group (OOH > OH) and positional isomer (14-OOH, 14-OH, 20-OOH > 11-OOH, 17-OOH > 10-OOH > 11-OH, 8-OOH, 7-OOH > 4-OOH).