Comparisons of the deduced amino acid sequences of eight primate photopigment genes led to the proposal that three amino acid substitutions produce the approximately 1,000 cm-1 difference in the absorption maxima of human red and green pigments (Neitz, M., Neitz, J., and Jacobs, G.H. (1991) Science 252, 971-974). We tested this proposal by mutating these three residues in rhodopsin and evaluating the effects on spectral properties. Nonpolar residues normally present in rhodopsin and in the green pigment were substituted by hydroxyl-bearing residues normally present in the red pigment. Two of these substitutions (Phe-261 to Tyr or Ala-269 to Thr) caused significant red shifts in the absorption maxima of the resulting mutant pigments. A third substitution (Ala-164 to Ser) caused only a slight effect. Combinations of substitutions caused additive shifts in absorption maxima. A double mutant (Phe-261 to Tyr/Ala-269 to Thr) displayed an absorption maximum that was red-shifted by 775 cm-1. Wavelength modulation in the visual pigments responsible for red-green color vision is likely to be governed by retinal-protein interactions involving primarily these two amino acid residues. Furthermore, interactions of hydroxyl-bearing amino acids with the chromophore may be a general mechanism of the opsin shift in visual pigments.