Cyclic GMP-dependent protein kinase, purified to homogeneity from bovine lung, was shown to activate hormone-sensitive lipase partially purified from chicken adipose tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher concentrations of the cyclic GMP-dependent enzyme were required (relative activities expressed in terms of histone H2b phosphorylation units). Activation by cyclic AMP-dependent protein kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the cyclic GMP enzyme was not inhibited. Lipase fully activated by cyclic AMP-dependent protein kinase showed no further change in activity when treated with cyclic GMP-dependent protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phosphorylase phosphatase (from bovine heart); full activity was restored by reincubation with cyclic GMP and cyclic GMP-dependent protein kinase. Cholesterol esterase activity in the chicken adipose tissue fraction, previously shown to be activated along with the triglyceride lipase by cyclic AMP-dependent protein kinase, was also activated by cyclic GMP-dependent protein kinase. Crude preparations of hormone-sensitive triglyceride lipase from human or rat adipose tissue and cholesterol esterase from rat adrenal were also activated by cyclic GMP-dependent protein kinase. Purified phosphorylase kinase (rabbit skeletal muscle) was also shown to be activated by cyclic GMP-dependent protein kinase. The present results, together with those of other workers on histone phosphorylation, suggest that the substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinase may be similar. This is discussed in the light of a model recently proposed with regard to the relationship between the subunit structures of the two kinases. The physiologic significance of the findings remains to be established.