Lysosomal acid lipase (LAL) is required for the hydrolysis of intracellular cholesteryl esters and triglycerides that are delivered to lysosomes by low density lipoprotein (LDL) receptor-mediated endocytosis. To understand that the expression of LAL mRNA and protein is tissue and cell specifically regulated in mice, genomic clones for the mouse lysosomal acid lipase (mLAL) gene were isolated and characterized. The 6.8 kb of the mLAL gene 5'-flanking region was sequenced. Comparisons of mouse and human LAL genes organization revealed identical intron/exon boundaries, except for intron 1 of the mouse gene, and identical exonic length of exons 3-9. The transcription start sites and exon 1 of mLAL were characterized by 5'-RACE-PCR and S1 nuclease mapping. Transfection of 5' flanking deletions of mLAL luciferase reporter gene construct identified positive and negative regulatory elements that varied with cell type. Transfection of three progressively smaller pieces of intron 1 inserted into an SV40 promoter and luciferase reporter gene revealed an enhancer-like activity in intron 1 that is also cell type specific. These studies provide insight into the basis for regulation of this critical enzyme in lipid metabolism.