To analyze the human red, green, and red-green hybrid cone pigments in vivo, we studied 41 male dichromats, each of whose X chromosome carries only a single visual pigment gene (single-gene dichromats). This simplified arrangement avoids the difficulties of complex opsin gene arrays and overlapping cone spectral sensitivities present in trichromats and of multiple genes encoding identical or nearly identical cone pigments in many dichromats. It thus allows for a straightforward correlation between each observer's spectral sensitivity measured at the cornea and the amino acid sequence of his visual pigment. For each of the 41 single-gene dichromats we determined the amino acid sequences of the X-linked cone pigment as deduced from its gene sequence. To correlate these sequences with spectral sensitivities in vivo, we determined the Rayleigh matches to different red/green ratios for 29 single-gene dichromats and measured psychophysically the spectral sensitivity of the remaining green (middle wavelength) or red (long wavelength) cones in 37 single-gene dichromats. Cone spectral sensitivity maxima obtained from subjects with identical visual pigment amino acid sequences show up to a approximately 3 nm variation from subject to subject, presumably because of a combination of inexact (or no) corrections for variation in preretinal absorption, variation in photopigment optical density, optical effects within the photoreceptor, and measurement error. This variation implies that spectral sensitivities must be averaged over multiple subjects with the same genotype to obtain representative values for a given pigment. The principal results of this study are that (1) approximately 54% of the single-gene protanopes (and approximately 19% of all protanopes) possess any one of several 5'red-3'green hybrid genes that encode anomalous pigments and that would be predicted to produce protanomaly if present in anomalous trichromats; (2) the alanine/serine polymorphism at position 180 in the red pigment gene produces a spectral shift of approximately 2.7 nm; (3) for each exon the set of amino acids normally associated with the red pigment produces spectral shifts to longer wavelengths, and the set of amino acids normally associated with the green pigment produces spectral shifts to shorter wavelengths; and (4) changes in exons 2, 3, 4, and 5 from green to red are associated with average spectral shifts to long wavelengths of approximately 1 nm (range, -0.5 to 2.5 nm), approximately 3.3 nm (range, -0.5 to 7 nm), approximately 2.8 nm (range, -0.5 to 6 nm), and approximately 24.9 nm (range, 22.2-27.6 nm).