Abstract  Background: Both β₁‐and β₂‐adrenoceptors (AR) in cardiac tissues are responsible for the excitatory effect of catecholamines. Recent evidence demonstrated the presence of another subtype ofβ‐AR (β₃‐AR) in cardiac ventricular preparation. Activation of β₃‐AR elicited a depressant response on ventricular contraction. The underlying mechanism(s) for the negative inotropism is relatively unknown. Methods: We investigated the effects of β₃‐AR activation on basal voltage‐dependent Ca²⁺ channel (l_CaL) amplitude of the guinea pig enzyme‐dissociated single ventricular myocytes using amphotericin B (200 μg/mL) perforated‐patch whole‐cell patch‐clamp techniques (˜ 22°C). Results: Application of (‐)‐isoprenaline ((‐)‐ISO) (100 nM, a nonselective β‐AR agonist) increased the basal l_CaL amplitude (˜ 210% of control) (n = 8). However, in the presence of nadolol (1 μM, a β₁‐/β₂‐AR antagonist), the stimulatory effect of (‐)‐ISO on l_CaL was abolished and a slowly developed inhibition of the basal l_CaL was recorded (˜ 80% of control) (n = 9). A smaller degree of inhibition was observed with BRL 37344 (100 nM, a selective β₃‐AR agonist) (58% of control) (n # 7). The inhibitory effect of (‐)‐ISO (with nadolol) and BRL 37344 persisted after washout. Pretreating the ventricular myocytes with L‐NAME (0.3 μM, a nitric oxide synthase inhibitor), but not D‐NAME (0.3 μM), abolished the inhibitory effect of (‐)‐ISO and BRL 37344 (n = 7–9). Conclusions: The results suggest that β₃‐ARs are present in ventricular myocytes. Activation of the β₃‐AR resulted in an inhibition of the basal l_CaL amplitude probably due to the formation of nitric oxide.