The catabolism of triglycerides-rich lipoproteins and the subsequent uptake of free fatty acids by muscle and adipose tissue is dependent on the enzyme lipoprotein lipase (LPL). To better understand the regulation of this enzyme, we have isolated and characterized the mouse LPL gene. The gene is 28 kb in length and comprises 10 exons which encode a 4.0-kb mRNA. In this report, almost 6 kb of DNA sequence is presented, including 1251 bp 5′ to the gene, over 4 kb of exon and exon-intron junctions, and 583 bp 3′ to the gene. RNA from differentiated 3T3-L1 adipocytes was used in primer extension and RNase protection assays to show that the 5′ untranslated region is not interrupted by an intron and the start site of transcription is 199 bp 5′ to the ATG codon that begins translation. The first exon codes for the 5′ untranslated region and the signal peptide of 27 amino acids and 2 amino acids of the mature protein, exons 2–9 code for 445 amino acids of the mature protein. These exons are short and vary in length from 102 to 287 bp. The 10th exon codes for the 3′ untranslated region and is 2346 bp long. This exon contains a single copy of a B1 repetitive element of 152 bp followed by a 169-bp homopurine stretch. These elements are flanked by a pair of 16-bp direct repeats. The mouse gene is similar in size to the human, which also contains 10 exons in similar locations. There is a high degree of sequence homology between the two genes, 5′ region (700 bp), 75%; 5′ untranslated region, 74%; coding region, 88%; 3′ untranslated region, 75%. The most striking difference is the absence of the B1 repetitive element and homopurine region in the human 3′ untranslated region. This information about the mouse LPL gene may lead to a better understanding of its regulation and role in plasma lipoprotein metabolism.