Concentration of adenosine 3', 5'-monophosphate (cAMP), binding activity of cAMP to protein and activities of cAMP-dependent and-independent protein kinase were determined in adrenal glands of rats treated with ACTH (Cortrosyn-Z). cAMP was increased rapidly after 6 U. ACTH injection, reached the maximum level (20-30 times the control) at 10min and returned to the control level by 120min. Hypertrophied adrenals of rats given repeated injections of 3 U. ACTH every 12hr contained a significantly higher amount of cAMP than untreated control. Treatment of animals with ACTH did not modify the activity of cAMP-binding protein throughout the experimental period. cAMP-independent activity of protein kinase was not influenced by ACTH but cAMP-dependent activity of the enzyme disappeared completely immediately after ACTH administration and a part of the activity was restored 80min after the treatment. Administration of cycloheximide prior to ACTH treatment did not disturb the effect of ACTH on cAMP formation and cAMP-dependent protein kinase activity.