Chloride ions are known to bind and alter the absorption spectra of some but not all visual pigments. In this report, the human red and green color vision pigments are shown to bind Cl- and to undergo a large red shift in their absorption maxima. Mutation of 18 different positively charged amino acids in these pigments identified two residues, His197 and Lys200, in the Cl(-)-binding site. His197 and Lys200 are strictly conserved in all long-wavelength cone pigments but are absent in all rhodopsins and short-wavelength cone pigments. This fact suggests that the evolutionary branch of the long-wavelength pigments was established when an ancestral pigment acquired the ability to bind Cl- and, as a result, shift the absorption maximum to longer wavelengths.