The roles of nitric oxide and of endothelium in the effects of the vasorelaxing agents acetylcholine and bradykinin on the production of prostanoids was studied in the isolated and perfused mesenteric vascular bed of the rat. Prostanoids were measured in the perfusate by high-performance liquid chromatography (HPLC). In the intact vascular bed, 1 microM bradykinin increased the release of 6-keto-prostaglandinF(1alpha) (stable metabolite of prostacyclin) and of prostaglandin E2 and 10 microM acetylcholine stimulated the efflux of prostacyclin only. In the de-endothelialized vascular bed, bradykinin increased the release of prostacyclin whereas acetylcholine increased the efflux of thromboxane. The inhibition of nitric oxide synthesis with 100 microM N(G)-nitro-L-arginine methyl ester prevented the effect of bradykinin but did not modify the effects of acetylcholine on prostanoid release. In addition, 100 microM L-arginine reversed the inhibitory effect of N(G)-nitro L-arginine methyl ester on bradykinin-stimulated prostaglandin production. It is concluded that acetylcholine and bradykinin stimulate prostanoid release in the rat mesenteric vascular bed with different patterns and through different mechanisms.