Rabbit anterior mesenteric-portal vein was shown to exhibit a positive correlation between relaxation and cyclic AMP levels using different concentrations of isoproterenol for varying exposure times. No consistent relationship was found between cyclic AMP and relaxation using a series of phosphodiesterase inhibitors (papaverine, SC2964, or RA233). In sodium-free (Tris-substituted) Krebs' solution, the addition of isoproterenol still increased, cyclic AMP levels, but there was no relaxation. Dibutyryl cyclic AMP was also ineffective in sodium-free solution. Under identical conditions, both relaxation and increases in cyclic AMP produced by papaverine and SC2964 were found to be independent of extracellular sodium. We conclude, therefore, that increased cyclic AMP may be involved in isoproterenol-induced relaxation possibly acting via increased Na+–Ca2+ exchange, but plays only a small role, if any, in smooth muscle relaxation produced by papaverine, RA233, or SC2964.