Protons participate in most biologically important reactions, as substrates, products, cofactors and modulators, and proton transport is an essential step in energy transduction. The dynamics of protonation reactions have been studied extensively in solution and in model systems involving lipid-water interfaces, but have never been resolved at the timescale of the elementary molecular event. Here we show that, under appropriate conditions, binding and unbinding reactions of single protons and deuterium ions to a single site on the L-type calcium channel can be resolved and the protonation and deprotonation rates quantified. The protonation rate constant considerably exceeds previous estimates obtained in simpler systems. The functional consequences of channel protonation is a threefold reduction of the channel conductance, independent of the applied voltage. The data are consistent with the presence of a single protonatable group with pK in the physiological pH range, close to the external mouth of the channel. The two conductance levels of the open channel might be explained by greatly differing local potentials associated with the protonated and deprotonated state of the group.