The calcium- and phospholipid-dependent protein kinase-C (PKC) is a critical enzyme of cellular signal transduction. In this report we studied calcium-dependent total PKC activity in eight adrenocortical carcinomas (group 1), nine adrenocortical adenomas (group 2), six hyperplasias (group 3), and five human normal adrenal tissues (group 4). The PKC activity assay was based on phosphorylation of a specific synthetic peptide from myelin basic protein. The specificity of the assay was confirmed by using an inhibitor peptide common to alpha-, beta-, and gamma-isoenzymes of PKC. The median value in group 1 was 1.15 pmol 32P/min.micrograms protein (range, 0.55-2.19), that in group 2 was 1.2 (range, 0.74-2.7), that in group 3 was 0.915 (range, 0.6-1.7), and that in group 4 was 1.22 (range, 0.6-3.95). The calcium-dependent total PKC activity was similar in the four groups studied. We did not find any correlation between urinary total cortisol, serum cortisol, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, and estradiol concentrations and PKC activity. These findings suggest that the calcium-dependent PKC activity is not elevated in adrenocortical tumors and is not a useful marker of adrenocortical malignancy.