In rings of rat aorta, cyclopiazonic acid (CPA), a selective inhibitor of the internal membrane Ca2+-pump ATPase, gradually initiated a concentration-dependent contraction which was much less in endothelium-intact than in endothelium-denuded rings. In phenylephrine-precontracted rings with intact endothelium, CPA, like acetylcholine, produced endothelium-dependent relaxations in a concentration-dependent manner. These were nearly abolished by methylene blue (MB) or NG-nitro-L-arginine methylester (L·NAME). This inhibitory effect of L·NAME was reversed by L·arginine but not by D-arginine, indicating that CPA induced a release of endothelium-derived relaxing factor (EDRF) from endothelial cells leading to smooth muscle relaxation. Concentration-dependent relaxations induced by sodium nitroprusside, which is thought to act by releasing nitric oxide, were not inhibited by L·NAME, but were inhibited similarly by MB in endothelium-intact and -denuded rings. These results indicate that CPA causes EDRF-release from endothelial cells and support the recent hypothesis that release of intracellular Ca from stores is the initiating factor in EDRF release, possibly allowing ongoing Ca entry to sustain rele