In previous studies we showed that arachidonate (AA) and the cyclic endoperoxide/thromboxane (Tx) A 2 mimic U46619-induced auto- and cross-desensitization of human platelets to either agonist. The desensitizing effect of U46619 is direct, whereas that of AA is mediated by a cyclooxygenase-dependent metabolite. Desensitization by AA and U46619 is suppressed by antagonists of the endoperoxide/Tx receptor sites. In the present investigation we demonstrated that eicosapentaenoic (EPA) and docosahexaenoic acid (DCHA) the major polyunsaturated fatty acids of fish oil suppressed TxB 2 formation and prevented platelet activation by AA and U46619. This inhibition required the presence of EPA or DCHA, since platelets pre-treated with these fatty acids and washed before testing responded as controls to the stimulating agents. At 0.1 and 0.3 mM respectively, DCHA and EPA behaved as reversible inhibitors of cyclooxygenase or Tx synthetase (inhibition of the effects of AA) and as endoperoxides/TxA 2 receptor antagonist (inhibition of the effects of U46619). Co-exposure of DCHA (0.1 mM) with AA or U46619 prevents auto- and cross-desensitization to AA and U46619. Platelets exposed to 0.3 mM DCHA and washed became refractory to stimulation by AA, but responded as controls to U46619. EPA (0.3 mM) was fully removed from platelets, which responded to AA and to U46619. EPA and DCHA antagonize endoperoxide/TxA 2 directly, and thus prevent the stimulation-dependent desensitization, and additionally, inhibit the cyclooxygenase activity required for desensitization.