Hormone-sensitive lipase (Lipe) catalyzes both the release lease of fatty acids from storage triglycerides in adipocytes and the liberation of cholesterol from cholesterol esters in steroidogenic tissues. Lipe activity is regulated in a tissue-, development- and hormone-specific fashion, the latter in large part by serine phosphorylation. We cloned and sequenced the Lipe gene from the 129Sv strain mouse, including 2.7 kb of the 5' nontranslated region. The primary transcript of the 129Sv Lipe locus spans 9.6 kb and contains 9 exons. We studied the curious hypervariable region immediately 5' to the regulatory serine residues by aligning the peptide and nucleic acid sequences of mouse, human, and rat Lipe. We propose that much of the variability is attributable to differences in the copy number of a 12-nucleotide repeat that shifts the intron 7 acceptor splice site. Introns 1 and 7 contain B1 elements, which in intron 7 are immediately adjacent to a tetranucleotide repeat. The mouse Lipe promoter region contains numerous potential binding motifs for factors implicated in adipose tissue expression and hormone responsiveness including adipocyte determination- and differentiation-dependent factor 1 (ADD1/SREBP1).