Here we have examined the voltage and pH dependence of unitary Slo3 channels and used analysis of current variance to define Slo3 unitary current properties over a broader range of voltages. Despite complexity in Slo3 channel openings that precludes simple definition of the unitary conductance, average current through single Slo3 channels varies linearly with voltage at positive activation potentials. Furthermore, the average Slo3 unitary current at a given activation potential does not change with pH. Consistent with macroscopic conductance estimates, the apparent open probability of Slo3 channel exhibits a pH-dependent maximum, with limiting values around 0.3 at the most elevated pH and voltage. Estimates of Slo3 conductance at negative potentials support a weaker intrinsic voltage dependence of gating than is observed for Slo1. For the pH-regulated Slo3 K+ channel, the dependence of macroscopic conductance on pH suggests that the pH-sensitive mechanism regulates gating in an allosteric manner qualitatively similar to regulation of Slo1 by Ca 2+. Together, the results support the view that the regulation of macroscopic Slo3 currents by pH reflects regulation of gating equilibria, and not a direct effect of pH on ion permeation. Specifically, both voltage and pH regulate a closed-open conformational change in a largely independent fashion.