Apart from the well-described K+ channel blocking effects in vascular smooth muscle cells, monovalent quaternary ammonium ions may also interact with endothelial cells in the endothelium-intact mammalian arteries. The present study was aimed to examine the effect of tetrabutylammonium ions on endothelium-dependent and -independent relaxation in the rat isolated aortic rings. Pretreatment with tetrabutylammonium concentration dependently reduced the endothelium-dependent relaxation induced by acetylcholine, cyclopiazonic acid and ionomycin. Tetrabutylammonium also inhibited endothelium-independent relaxation induced by hydroxylamine or nitroprusside. Pretreatment of endothelium-denuded rings with tetrabutylammonium did not affect relaxation induced by NS1619 or by diltiazem. In contrast, tetrabutylammonium significantly reduced the pinacidil- or cromakalim-induced relaxation. Tetrabutylammonium also inhibited the acetylcholine- but not nitroprusside-induced increase of tissue content of cyclic GMP in the aortic rings. The present study indicates that tetrabutylammonium ions could inhibit endothelial and exogenous nitric oxide-mediated aortic relaxation while it had no effect on relaxation induced by activation of Ca2+-activated K+ channels (by NS1619) or by inhibition of voltage-gated Ca2+ channels (by diltiazem). The inhibitory effect on pinacidil- and cromakalim-induced relaxation suggests that tetrabutylammonium ions also inhibit ATP-sensitive K+ channels in aortic smooth muscle cells.