Hormone-sensitive lipase, detergent-solubilized and purified from rat adipose tissue, was phosphorylated with the catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Maximally 1.05 +/- 0.05 (mean +/- S.E. (n = 3) ) mol of phosphate/mol of hormone-sensitive lipase Mr = 84,000 subunit was incorporated. Phosphoserine was the only phosphorylated amino acid residue. A single phosphorylation site was demonstrated by digestion with Staphylococcus aureus V8 protease and trypsin that produced a single acidic phosphopeptide of about 10 amino acid residues length, which was isolated by two-dimensional electrophoresis-thin layer chromatography. Enzyme activity was enhanced, 2.5-fold against trioleoylglycerol, concomitant with phosphorylation, with half-maximal effect within 30 sec, a rate of phosphorylation of the enzyme comparable to that obtained in vivo (Nilsson, N. O., Strålfors, P., Fredrikson, G., and Belfrage, P. (1980) FEBS Lett. 111, 125-130). The initial rate of phosphorylation was approximately half that with phosphorylase kinase as substrate. The effects of modifications of hormone-sensitive lipase and of various additions and variation in pH were examined.