Phosphorylation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase results in activation of both cholesterol ester and triacylglycerol hydrolase activities. Activation against both substrates correlates closely with phosphorylation in time course experiments. Proteolytic digestion of phosphorylated cholesterol ester hydrolase, followed by peptide mapping, indicates the presence of a single phosphorylation site on the enzyme. Phosphoserine is the only phosphoamino acid detected following partial acid hydrolysis of the phosphorylated enzyme.