Effect of azide on proton transfer of bacteriorhodopsin (bR) mutants that lack the proton donor Asp-96 and/or proton acceptor Asp-85 was investigated by a photoelectrochemical method. In the presence of azide, D96N completely recovered the response profile of the wild type and D96N/D85N gave a very small current with the same amplitude as in D85N. On the contrary, D85N showed no azide effect. These results indicate that azide affects the process of shuttling protons from D96 to the Schiff base rather than does the proton transfer from the Schiff base to D85.