Protein kinase activity has been studied in four human adrenocortical tumors and compared to the one of the normal human adrenal. In two cases where the lack of action of ACTH was related to an anomaly of ACTH receptor, the protein kinase activity was normal. In the other two cases the ACTH receptor was normal, but the protein kinase activity was different from that of the normal adrenal. In one of these cases where the steroidogenesis response of isolated tumor cells to ACTH and DcAMP was higher than in normal adrenal, basal and cAMP stimulated protein kinase activities were significantly higher than those of the normal adrenal, but the activation constants of both nucleotides were similar to those of the normal gland. In the other case, the basal and the cAMP stimulated protein kinase activities were significantly lower, as well as the activation constant of cAMP. However, the binding affinity of 3H-cAMP was normal. Normal adrenal cytosol contains three protein kinases, as resolved by DEAE-cellulose, two of which designated I and II, are cAMP-dependent. The DEAE-cellulose chromatography of the last tumor showed a loss of isoenzyme II. In addition, the protein kinase eluted at the same molarity as that of isoenzyme I of the normal adrenal was not activated by cAMP. Therefore, the lack of response to ACTH of some adrenocortical human tumors may be attributed either to an anomaly of the ACTH receptor or to some defect of the cAMP-dependent protein kinase.