The gene locus for bovine IRBP, as well as several kilobases of sequence flanking the gene on either end, has been cloned. Two of the several clones seem to contain full-length copies of the protein encoding portion of the gene. Using these clones and cDNA clones, we have determined that there are one or perhaps two copies of the IRBP gene per haploid genome in several species. The gene is compact considering the large size of the protein (145,000 daltons) and its large mRNA (about 6.5 kb). Surprisingly, the gene is no more than 14 kb, being fully contained on lambda clones of maximum packaging size 20 kb. Small parts of the gene were sequenced for the purpose of proving the identity of the genomic clones. DNA sequencing of one of the IRBP gene clones demonstrates the existence of an intron in the gene. The sequence analysis of another fragment identified the N-terminus which has been sequenced at the protein level. The DNA sequence analysis showed the existence of a putative signal sequence and the potential existence of a short five amino acid sequence between the signal sequence and the authentic N-terminus of the secreted extracellular IRBP. This confirms and validates the finding of the extra five amino acid sequence that is present on 40-50% of the polypeptides in monkey and human IRBP which have been isolated from the subretinal space. The presence of the appropriate gene sequence for the pentapeptide but its absence in bovine IRBP indicates differences in processing among the vertebrate IRBPs.