We have determined the nucleotide sequence of the gene encoding rat liver fatty acid-binding protein (L-FABP). Previous structural studies have shown that L-FABP belongs to a family of low molecular weight cytosolic proteins which bind hydrophobic ligands. Rat L-FABP is the first member of this family whose genomic organization has been defined. The L-FABP transcription unit spans 3790 nucleotides and contains four exons (115, 173, 93, and 121 base pairs) interrupted by three introns (1454, 1224, and 610 base pairs). No other abundant mRNAs appear to be transcribed from sequences located within 4 kilobases 5‘ or 6.5 kilobases 3‘ of this gene. Sequence analyses have detected the presence of several related amino acid sequence blocks within L-FABP which may serve similar structural roles. A variety of computational techniques were used to compare the oligopeptides specified by each exon with other known members of the protein family. The results indicate that only the amino acid sequence present in the first exon is conserved among the homologous proteins. This suggests that the first exon may encode a shared structural and functional domain important in the interaction of these proteins with their ligands.