We report the direct measurement of photoinduced surface potential differences of wild-type (WT) and mutant D96N bacteriorhodopsin ( BR) membranes at pH 7 and 10.5. Atomic force microscopy (AFM) and scanning surface potential microscopy (SSPM) were used to measure the BR membrane with the extracellular side facing up. We present AFM and SSPM images of WT and mutant D96N in which the light-dark transition occurred in the mid-scan of a single BR membrane. Photosteady-state populations of the M state were generated to facilitate measurement in each sample. The photoinduced surface potential of D96N is 63 mV (peak to valley) at pH 10.5 and is 48 mV at pH 7. The photoinduced surface potential of WT is 37 mV at pH 10.5 and similar to 0 at pH 7. Signal magnitudes are proportional to the amount of M produced at each pH. The results indicated that the surface potentials were generated by photoformation of surface charges on the extracellular side of the membrane. Higher surface potential correlated with a longer lifetime of the charges. A mechanistic basis for these signals is proposed, and it is concluded that they represent a steady-state measurement of the B2 photovoltage.