Bacteriorhodopsin is the light-activated proton pump in the purple membrane of Halobacterium salinarium. One aspect of the proton pump mechanism that remains to be fully revealed is how back-translocation of the proton is eliminated despite the fact that gradients of 250 mV or more can exist in favor of reverse proton motion. This mechanistic component is examined with reference to the static and photodynamic resonances observed in the 1-40 GHz region of the microwave spectrum, The light-adapted form of bacteriorhodopsin displays strong microwave absorption with maxima near 8 and 25 GHz. We identify the origin of the microwave activity to protein-bound divalent cations by noting that (1) the ion-free blue membrane is microwave inactive, and (2) the microwave activity of the protein can be regenerated by adding calcium and/or magnesium to the protein. Initiation of the photocycle generates additional microwave activity in the low-frequency region of the spectrum. The M minus bR difference spectrum shows a single-cation resonance at 9.8 GHz. We assign this resonance to the release of a calcium ion from the chromophore-adjacent binding site into the proton channel. We conclude that a key biological role of the chromophore-adjacent calcium binding site is to provide a divalent cation gate that not only facilitates chromophore reprotonation but also prevents back-transfer of the proton during the latter stages of the photocycle.