Precise regulation of N-type (Ca(V)2.2) voltage-gated calcium channels (Ca-channels) controls many cellular functions including neurotransmitter and hormone release. One important mechanism that inhibits Ca2+ entry involves binding of G-protein betagamma subunits (Gbetagamma) to the Ca-channels. This shifts the Ca-channels from "willing" to "reluctant" gating states and slows activation. Voltage-dependent reversal of the inhibition (facilitation) is thought to reflect transient dissociation of Gbetagamma from the Ca-channels and can occur during high-frequency bursts of action potential-like waveforms (APW). Inactivation of Ca-channels will also limit Ca2+ entry, but it remains unclear whether G-proteins can modulate inactivation. In part this is because of the complex nature of inactivation, and because facilitation of Ca-channel currents (I(Ca)) masks the extent and kinetics of inactivation during typical stimulation protocols. We used low-frequency trains of APW to activate I(Ca). This more closely mimics physiological stimuli and circumvents the problem of facilitation which does not occur at < or = 5 Hz. Activation of endogenous G-proteins reduced both Ca2+-dependent, and voltage-dependent inactivation of recombinant I(Ca) in human embryonic kidney 293 cells. This was mimicked by expression of wild-type Gbetagamma, but not by a point mutant of Gbetagamma with reduced affinity for Ca-channels. A similar decrease in the inactivation of I(Ca) was produced by P2Y receptors in adrenal chromaffin cells. Overall, our data identify and characterize a novel effect of G-proteins on I(Ca), and could have important implications for understanding how G-protein-coupled receptors control Ca2+ entry and Ca2+-dependent events such as neurotransmitter and hormone release.