Some blockers of β₁- and β₂-adrenoceptors cause cardiostimulant effects through an atypical β-adrenoceptor (putative β₄-adrenoceptor) that resembles the β₃-adrenoceptor. It is likely but not proven that the putative β₄-adrenoceptor is genetically distinct from the β₃-adrenoceptor. We therefore investigated whether or not the cardiac atypical β-adrenoceptor could mediate agonist effects in mice lacking a functional β₃-adrenoceptor gene (β₃KO). (−)-CGP 12177, a β₁- and β₂-adrenoceptor blocker that causes agonist effects through both β₃-adrenoceptors and cardiac putative β₄-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and β₃KO mice. The effects of (−)-CGP 12177 were resistant to blockade by (−)-propranolol (200 nm) but were blocked by (−)-bupranolol (1 μm) with an equilibrium dissociation constant of 15 nm in WT and 17 nmin β₃KO. (−)-[³H]CGP 12177 labeled a similar density of the putative β₄-adrenoceptor in ventricular membranes from the hearts of both WT (B _max = 52 fmol/mg protein) and β₃KO (B _max = 53 fmol/mg protein) mice. The affinity of (−)-[³H]CGP 12177 for the cardiac putative β₄-adrenoceptor was not different between WT (K _d = 46 nm) and β₃KO (K _d = 40 nm). These results provide definitive evidence that the cardiac putative β₄-adrenoceptor is distinct from the β₃-adrenoceptor.