Effects of activation of ₃-adrenoceptor have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of CL 316243, a selective ₃-adrenoceptor (₃-AR) agonist, on the basal smooth muscle tone, and direct release of nitric oxide by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a ₃-adrenoceptor antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a non-selective NOS inhibitor L-NNA, and by putative inhibitor of eNOS (L-NIO). Inhibitors of iNOS (1400W) and nNOS (TRIM) had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by L-NNA and L-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by ₃-AR activation, that is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal transduction pathways including NOS activation may explain the characteristic IAS relaxation by ₃-AR activation. The studies may have therapeutic implications in anorectal motility disorders.