β₃-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). β₃-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca²⁺ channels in normal hearts; however, the effects of β₃-AR activation on Ca²⁺ channel in HF remain unknown. We compared the effects of β₃-AR activation on L-type Ca²⁺ current (I_Ca,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). I_Ca,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a β₃-AR agonist, caused a dose-dependent decrease in I_Ca,L with maximal inhibition (21%, 1.1 ± 0.2 versus 1.4 ± 0.1 nA) (p < 0.01) at 10^–7 M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in I_Ca,L (1.1 ± 0.2 versus 1.6 ± 0.2 nA) (p < 0.05). A similar inhibition of I_Ca,L was also observed with ISO (10^–7 M) in the presence of a β₁- and β₂-AR antagonist, nadolol (10^–5 M). Inhibition was abolished by the β₃-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10^–6 M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N^G-nitro-l-arginine methyl ester (10^–4 M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 μg/ml, 36°C, 6 h). In conclusion, β₃-AR activation inhibits L-type Ca²⁺ channel in both normal and HF myocytes. In HF, β₃-AR stimulation-induced inhibition of Ca²⁺ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.