MthK is a calcium-gated, inwardly rectifying, prokaryotic potassium channel. Although little functional information is available for MthK, its high-resolution structure is used as a model for eukaryotic Ca(2+)-dependent potassium channels. Here we characterize in detail the main gating characteristics of MthK at the single-channel level with special focus on the mechanism of Ca(2+) activation. MthK has two distinct gating modes: slow gating affected mainly by Ca(2+) and fast gating affected by voltage. Millimolar Ca(2+) increases MthK open probability over 100-fold by mainly increasing the frequency of channel opening while leaving the opening durations unchanged. The Ca(2+) dose-response curve displays an unusually high Hill coefficient (n = approximately 8), suggesting strong coupling between Ca(2+) binding and channel opening. Depolarization affects both the fast gate by dramatically reducing the fast flickers, and to a lesser extent, the slow gate, by increasing MthK open probability. We were able to capture the mechanistic features of MthK with a modified MWC model.