We previously showed that activation of G(i/o)-coupled histamine H(3)-receptors (H(3)R) is cardioprotective because it attenuates excessive norepinephrine release from cardiac sympathetic nerves. This action is characterized by a marked decrease in intraneuronal Ca(2+) ([Ca(2+)](i)), as G alpha(i) impairs the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, and this decreases Ca(2+) influx via voltage-operated Ca(2+) channels (VOCC). Yet, the G(i/o)-derived betagamma dimer could directly inhibit VOCC, and the subsequent reduction in Ca(2+) influx would be responsible for the H(3)R-mediated attenuation of transmitter exocytosis. In this study, we tested this hypothesis in nerve-growth factor-differentiated rat pheochromocytoma cells (PC12) stably transfected with H(3)R (PC12-H(3)) and with the G betagamma scavenger beta-adrenergic receptor kinase 1 (beta-ARK1)-(495-689)-polypeptide (PC12-H(3)/beta-ARK1). Thus, we evaluated the effects of H(3)R activation directly on the following: 1) Ca(2+) current (I(Ca)) using the whole-cell patch-clamp technique; and 2) K(+)-induced exocytosis of endogenous dopamine. H(3)R activation attenuated both peak I(Ca) and dopamine exocytosis in PC12-H(3) but not in PC12-H(3)/beta-ARK1 cells. Moreover, a membrane permeable phosducin-like G betagamma scavenger also prevented the antiexocytotic effect of H(3)R activation. In contrast, the H(3)R-induced attenuation of cAMP accumulation and dopamine exocytosis in response to forskolin were the same in both PC12-H(3) and PC12-H(3)/beta-ARK1 cells. Our findings reveal that although G alpha(i) participates in the H(3)-mediated antiexocytotic effect when the adenylyl cyclase-cAMP-PKA pathway is stimulated, a direct G betagamma-induced inhibition of VOCC, resulting in an attenuation of I(Ca), plays a pivotal role in the H(3)R-mediated decrease in [Ca(2+)](i) and associated cardioprotective antiexocytotic effects. The discovery of this H(3)R-signaling step may offer new therapeutic approaches to cardiovascular diseases characterized by hyperadrenergic activity.