Rat genomic DNA fragments bearing the retinol-binding protein (RBP) gene have been isolated and characterized. The gene spans 6.9 kilobases and contains six exons. The five intervening sequences range in size from 78 base pairs to 4.4 kilobase pairs with the first interrupting the 5' untranslated region. A comparison of the gene organization with the three-dimensional structure of RBP reveals that all translated exon transcripts closely correspond to discrete tertiary structural elements. Residues of the protein involved in the retinol binding are encoded by three separate exons. It has been proposed that the two regions displaying internal homology in the human RBP, both at the primary and tertiary structure levels, arose by a partial ancestral gene duplication. If such an event were involved, evidence for it at the nucleotide sequence and exon-intron organization levels has been obscured.