Although β 3-adrenoceptors (ARs) have been extensively characterized in brown and white adipocytes, their actions in the beating heart are unclear. We examined the effects of a β 3-AR agonist, BRL37344, on cardiac function and calcium transients in Langendorff-perfused guinea pig hearts by simultaneously measuring left ventricular (LV) pressure and Ca 2+-dependent indo-1 fluorescence. BRL-37344 induced a dose-dependent negative inotropic effect at concentrations from 10− 11 to 10− 8 M. Maximally, LV developed pressure decreased to 80±2%,+ dP/dt to 81±2%, and− dP/dt to 81±3% of their respective control values (p< 0.01). The amplitude of the Ca 2+ transient also decreased (to 92±3% of the control level; p< 0.01). The BRL37344 dose-response curve was not altered by nadolol (10− 5 M), a potent β 1-and β 2-AR antagonist, but completely suppressed by bupranolol (10− 6 M), a potent β 1-, β 2-and β 3-AR antagonist. To assess the potential role of a nitric oxide synthase (NOS) pathway, we determined whether the NOS inhibitor, N G-nitro-L-arginine methyl ester (L-NAME), modified the contractile response to BRL37344. L-NAME (10− 7 and 10− 4 M) attenuated the negative inotropic effects on LV developed pressure by 35 and 50%, suggesting that β 3-AR stimulation induces a negative inotropic effect on guinea pig hearts partly through a decrease in the Ca 2+ transient and partly by the NOS pathway.