A cloned 2184-bp cDNA coding for human interstitial retinol-binding protein (IRBP) has been isolated and sequenced. The probe hybridized to a 5.2-kb poly(A) RNA from human retinas. Nineteen tryptic peptides (363 amino acids) sequenced and purified from bovine IRBP could be aligned with 86-88% homology to the translated sequence. Two segments approximately 200 amino acids long were found to have a 41% residue identity, suggesting an internal duplication event. This cloned cDNA was used to probe DNA samples from a panel of 29 rodent-human somatic cell hybrids, mapping the structural gene for IRBP to chromosome 10. In situ hybridization suggested a regional localization near the centromere (p11.2----q11.2), although a secondary site of hybridization at q24----25 was also observed.