Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase whose activity is regulated by reversible phosphorylation and which is thought to be the rate-limiting enzyme for the mobilization of FFA from adipose tissue. In the current studies the subunit structure of HSL has been explored using sucrose gradient centrifugation and in vivo and in vitro protein-protein interactions. Evidence is provided to demonstrate that HSL exists as a functional dimer composed of homologous subunits. Dimeric HSL displayed approximately 40-fold greater activity against cholesteryl ester substrate when compared with monomeric HSL without any differences in affinity for the substrate. Truncations of HSL identified the importance of the N-terminal 300 amino acids, as well as other regions, in participating in the oligomerization of HSL. These studies support the notion that the N-terminal region of HSL represents a docking domain for protein-protein interactions and provide an additional mechanism for the posttranslational control of HSL activity in the cell via oligomerization.