The effect of sulfonylureas on contractility in atria isolated from rabbits, rats, and guinea pigs was examined. Tolbutamide was most efficacious on rabbit preparations, and 1 mM tolbutamide increased tension development about 30 per cent above control. Tolbutamide did not elevate cyclic adenosine 3',5'-monophosphate (cyclic AMP) or increase the rate of spontaneously contracting rabbit, rat, or guinea pig preparations. Glibenclamide, which also increased tension development, was about 300 times as potent as tolbutamide, paralleling its potency relative to tolbutamide as an insulinotropic agent. Tolbutamide enhanced adenylate cyclase activity in homogenates of rabbit myocardium only at very high concentrations (1 to 10 mM), and this activation was attributable to a nonspecific detergent action. Glibenclamide, at concentrations as high as 100 μM, failed to activate adenylate cyclase. Three inotropic agents, thought to act by elevating myocardial cyclic AMP—norepinephrine, histamine, and the phosphodiesterase inhibitor l-methyl-3-isobutyl xanthine (MIX)—failed to increase tension developed when added after a maximally effective concentration of isoproterenol. However, both calcium ion and tolbutamide could increase systolic and diastolic tension when added to contracting atria after maximal isoproterenol. It is concluded that the inotropic action of sulfonylureas is not mediated by cyclic AMP and may result from an effect upon myocardial calcium metabolism.