Human cDNA encoding OPN1LW was inserted at the endogenous mouse Opn1mw locus, replacing a 3' portion of exon 2 and all of exons 3, 4, and 5. The allele putatively produces a hybrid protein consisting of 92 N-terminal residues encoded by mouse exon 1 and the 5' portion of exon 2, and 266 C-terminal residues encoded by the human cDNA. Nearly the entire chromophore binding pocket, as well as the residues that account for the difference in absorbance spectra between human and mouse, is derived from the human sequence. Immunostaining indicated expression of the hybrid protein and electroretinogram indicated that the spectral sensitivity of cones containing the targeted allele was similar to that of human red cones.