Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.