A unique glutamic acid-rich protein was previously identified in bovine rod photoreceptors (Sugimoto et al., 1991, Proc. Natl. Acad. Sci. USA 88: 3116-3119) and later suggested to be a third subunit (gamma) of the rod cGMP-gated cation channel (Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11757-11761). Here, we report on the characterization of the GAR1 gene encoding a human homolog of bovine gamma. Sequence analysis of cDNA clones encoding human gamma revealed an open reading frame predicting a protein of 299 amino acids (approximately 32 kDa), half the size of the bovine gamma subunit. Comparison of the N-terminal half of bovine gamma with the predicted human gamma sequence revealed 90% identity within the first 31 amino acids, and only 60% homology was found throughout the remainder of the protein sequence. As in bovine gamma, the predicted isoelectric point of the human protein is very acidic despite the absence of the bovine C-terminal glutamic acid-rich domain. The integrity of the cDNA sequence was confirmed by analysis of several overlapping genomic clones that span the GAR1 gene. The protein coding region of the gene consists of 12 exons spanning approximately 11 kb with exon sequence identical to that of the cDNA clones. PCR of somatic cell hybrid DNA with primer pairs that amplify a portion of the GAR1 gene (locus designation CNCG3L) demonstrate localization to chromosome 16. The location of the gene was further delimited by fluorescence in situ hybridization placing the gene at 16q13.(ABSTRACT TRUNCATED AT 250 WORDS)