The keratinocyte lipid-binding protein (KLBP) is a member of a large multigene family of intracellular fatty-acid-binding proteins. It is expressed in skin and tongue epithelia, adipose, lung and mammary tissue and has been found upregulated in several skin cell carcinomas and papillomas (Krieg et al., 1993). In order to study the regulation of KLBP expression, the murine gene has been cloned. Southern analysis using an exon 2 specific cDNA probe indicated the presence of multiple copies of the gene in the murine genome. Based on the highly conserved structure of the fatty-acid-binding protein genes, the third intron of the KLBP gene was PCR-amplified utilizing murine genomic DNA. Southern analysis with the intron 3 probe identified one unique gene in the murine genome. A full-length genomic clone of KLBP was obtained from a P1 library, and the structural gene was sequenced. Similar to the other FABP genes, the functional KLBP gene contains four exons separated by three introns and maintains the conservation of size and placement of each exon. A functional minimal promoter was demonstrated by transient transfections of 5' upstream KLBP-luciferase reporter constructs into line 308 keratinocyte cells as well as in primary adipocytes. RT-PCR on primary adipocyte RNA demonstrated expression of this KLBP gene by amplification of intron 3 from the primary transcript. Fluorescence in-situ hybridization identified the murine KLBP gene as the fourth FABP gene on chromosome 3, along with myelin P2, ALBP, and intestinal FABP. These studies provide a framework for analysis of KLBP expression in normal and pathophysiological conditions.