Assignment of the protonation state of the residue Glu-181 is important to our understanding of the primary event, activation processes and wavelength selection in rhodopsin. Despite extensive study, there is no general agreement on the protonation state of this residue in the literature. Electronic assignment is complicated by the location of Glu-181 near the nodal point in the electrostatic charge shift that accompanies excitation of the chromophore into the low-lying, strongly allowed pi pi* state. Thus, the charge on this residue is effectively hidden from electronic spectroscopy. This situation is resolved in bathorhodopsin, because photoisomerization of the chromophore places Glu-181 well within the region of negative charge shift following excitation. We demonstrate that Glu-181 is negatively charged in bathorhodopsin on the basis of the shift in the batho absorption maxima at 10K [lambda(max) band (native) = 544 +/- 2 nm, lambda(max) band (E181Q) = 556 +/- 3 nm] and the decrease in the lambda(max) band oscillator strength (0.069 +/- 0.004) of E181Q relative to that of the native protein. Because the primary event in rhodopsin does not include a proton translocation or disruption of the hydrogen-bonding network within the binding pocket, we may conclude that the Glu-181 residue in rhodopsin is also charged.