The Ca2+ channel beta subunit has been shown to reduce the magnitude of G-protein inhibition of Ca2+ channels. However, neither the specificity of this action to different forms of G-protein inhibition nor the mechanism underlying this reduction in response is known. We have reported previously that coexpression of the Ca2+ channel beta3 subunit causes M2 muscarinic receptor-mediated inhibition of alpha1B Ca2+ currents to become more voltage-dependent. We report here that the beta3 subunit increases the rate of relief of inhibition produced by a depolarizing prepulse and also shifts the voltage dependency of this relief to more hyperpolarized voltages; these effects are likely to be responsible for the reduction of inhibitory response of alpha1B channels to G-protein-mediated inhibition seen after coexpression of the Ca2+ channel beta3 subunit. Additionally, the beta3 subunit alters the rate and voltage dependency of relief of the inhibition produced by coexpressed Gbeta1gamma1, in a manner similar to the changes it produces in relief of M2 receptor-induced inhibition. We conclude that the Ca2+ channel beta3 subunit reduces the magnitude of G-protein inhibition of alpha1B Ca2+ channels by enhancing the rate of dissociation of the G-protein betagamma subunit from the Ca2+ channel alpha1B subunit.