Dietary enrichment of membrane phospholipids with n-3 (fish-oil-derived) fatty acids has attracted attention as a putative therapeutic regimen for suppression of inflammatory and coagulatory events. Use of n-3 fatty-acid-enriched lipid infusions for parenteral nutrition results in micromolar concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) in the plasma-free fatty acid fraction. We investigated the influence of free EPA and DCHA on platelet thromboxane (Tx) A2 and A3 formation by using a recently developed high performance liquid chromatography-ELISA technique for separate quantification of the stable hydrolysis products TxB2 and TxB3. Washed human thrombocytes were incubated with free arachidonic acid (AA; 1 microM), A23187 (0.1 microM) or thrombin (5 U/mL) for stimulation; all regimens provoked large quantities of TxA2 in the absence of TxA3. Simultaneous admixture of free EPA or free DCHA to the incubation medium (concentration range, 0.01-50 microM) largely suppressed platelet TxA2 generation in response to all stimuli used in a dose-dependent manner. The effective concentration with 50% influence of arachidonic acid was 4.2 microM, whereas the inhibitory concentration with 50% effect of EPA and DCHA were both in the same order of magnitude but differed with the nature of the agonist (0.2-7 microM). Platelet (co-)incubation with EPA, but not DCHA, provoked dose-dependent synthesis of n-3-lipid-derived thromboxane: kinetics of formation and absolute quantities of TxA3 approximated 20% of the respective TxA2 data upon stimulation with AA. Both EPA and DCHA dose-dependently suppressed U46619-provoked platelet aggregation. We conclude that EPA and DCHA are potent competitive inhibitors of TxA2 generation by intact platelets, with EPA acting as poor substrate and DCHA being no substrate for the cyclooxygenase/thromboxane synthase complex. Enrichment of the plasma-free fatty acid fraction with n-3 lipids may offer a therapeutic regimen to suppress the synthesis of the potent proaggregatory and vasoconstrictory agent TxA2.