A partially purified hormone-sensitive triglyceride lipase of human adipose tissue was found to be activated twofold by the addition of cyclic 3′,5′-AMP, ATP, and magnesium ions. Lipase activities against diolein and monoolein were not affected. Addition of protein kinase inhibitor at zero time completely inhibited activation, and this inhibition was prevented by prior addition of an excess of exogenous protein kinase (from rabbit skeletal muscle). Addition of protein kinase inhibitor during the activation step blocked the activation process without a time lag, suggesting that protein kinase operates directly on hormone-sensitive lipase. Further purification yielded a fraction free of protein kinase, and lipase activation in this fraction depended absolutely on addition of exogenous kinase. Incubation of human fat with epinephrine or isoproterenol stimulated lipolysis and caused conversion of nonactivated hormone-sensitive lipase to its activated form, as indicated by a decrease in the activation subsequently obtainable in fractions prepared from such hormone-treated tissues. These findings strongly suggest that the stimulation of lipolysis by hormonal treatment is the consequence of the activation of hormone-sensitive triglyceride lipase by cyclic 3′,5′-AMP-dependent protein kinase.