Hormone-sensitive lipase (HSL) is a key enzyme regulating the acute activation of lipolysis. HSL functionality is controlled by multiple phosphorylation events, which regulate its association with the surface of lipid droplets (LDs). We determined the progression and stability of HSL phosphorylation on individual serine residues both spatially and temporally in adipocytes using phospho-specific antibodies. Within seconds of beta-adrenergic receptor activation, HSL was phosphorylated on Ser-660, the phosphorylated form appearing in the peripheral cytosol prior to rapid translocation to, and stable association with, LDs. In contrast, phosphorylation of HSL on Ser-563 was delayed, the phosphorylated protein was predominantly detected on LDs, and mutation of the Ser-659/Ser-660 site to Ala significantly reduced subsequent phosphorylation on Ser-563. Phosphorylation of HSL on Ser-565 was observed in control cells; the phosphorylated protein was translocated to LDs with similar kinetics to total HSL, and the degree of phosphorylation was inversely related to phospho-HSL(Ser-563). These results describe the remarkably rapid, sequential phosphorylation of specific serine residues in HSL at spatially distinct intracellular locales, providing new insight into the complex regulation of lipolysis.