We have investigated the effect of permeant ion species on activation of transiently expressed neuronal alpha1A Ca channels. Equimolar replacement of Ba with Ca resulted in a consistent depolarizing shift of the half-activation potential whose magnitude (∼10 mV) was constant over a range of 2 to 100 mM permeant ion, suggesting that the effects of Ca ions were fully developed at concentrations below 2 mM and indicating that Ba and Ca screened surface charges equally. In mixtures of Ba and Ca at constant divalent cation concentration the voltage-shift, as a function of Ca mole fraction, was well described by a model in which Ba and Ca compete for a single site but only Ca ions produce a gating effect. Overall, our data are consistent with Ca ions exerting their effects on activation via a specific regulatory site.