Many of the intracellular actions of cyclic adenosine 3',5'-monophosphate are expressed through phosphorylation reactions mediated by cAMP-dependent protein kinases, but little is known about hormonal control of endogenous protein kinase activity (PK) in kidney. In the present study, we examined the effects of parathyroid hormone, glucagon, and isoproterenol on cAMP and PK in slices of rat renal cortex. In the presence of 0.5 mM 1-methyl, 3-isobutyl xanthine, all three hormones activated PK in slices, as reflected by an increase in the ratio of enzyme activity assayable in homogenates of the slices without addition of cAMP to the kinase reaction mixture (cAMP-independent activity) over total enzyme activity (+2 uM cAMP in the reaction mixture). When enzyme activity was assayed in whole homogenates prepared from slices, the increase in the enzyme activity ratio (- cAMP/+cAMP) which followed hormonal stimulation was due entirely to an increase in cAMP-independent activity, with no change in total activity. In general, a good correlation existed between the alterations in tissue cAMP levels mediated by the hormones and/or 1-methyl, 3-isobutyl xanthine and concomitant alterations in PK. All three hormones increased PK activity ratios to near unity, suggesting complete enzyme activation. However, the concentrations of parathyroid hormone and glucagon which produced maximal activation of PK were much lower than those required for maximal cAMP responses. Studies with charcoal indicated that these hormonal actions on PK reflected intracellular events rather than representing activation of the enzyme during tissue homogenization, due to release of sequestered cAMP. Thus, homogenization of tissue in charcoal prevented activation of PK by subsequent addition of exogenous cAMP, but did not lower enzyme activity ratios in homogenates of hormone-stimulated cortical slices. When PK was determined in the 20,000 g supernatant fraction of renal cortical slices incubated with the hormones, enzyme activity ratios also increased, but total enzyme activity declined. Lost activity was recovered by extraction of particulate fractions with 500 mM KCl or NaCl, results which implied particulate binding of activated PK. Activated soluble PK from renal cortex was bound equally well by intact, heat- and trypsin-treated renal cortical pellets and by intact and heated hepatic pellets. Accordingly, the apparent translocation of enzyme in hormone stimulated cortex does not necessarily represent binding of the activated PK to specific acceptor sites in the particulate cell fractions or constitute a physiologic hormonal action. Activation of renal cortical PK by increasing concentrations of salts suggests that the enzyme in this tissue resembles the predominant type found in heart.