In vivo fate of a threefold substituted ACTH 4-9 analog with a markedly potentiated behavioral activity, 4-Met(O2), 8-D-Lys, 9-Phe-ACTH 4-9, was investigated. The radioactive labeled [7-3H-Phe] ACTH 4-9 analog was administered IV, SC and orally in a dose of approximately 40 mug. Plasma concentrations of total radioactivity and intact peptide were determined at various periods after administration in urethane anesthetized rats. Oral administration was also performed with conscious animals. Maximal plasma concentrations were found 8 min after SC injection. After oral administration in anesthetized rats maximal plasma levels were reached 8 hr after administration; in conscious animals this took 4 hr. The initial volume of distribution was 5.9% of body weight and the initial half-life (t1/2) for intact peptide 4 min. Shortly after IV and SC administration relatively high and stable plasma levels of intact peptide were obtained, reflecting metabolic stability. This stability was also apparent from the metabolite patterns, which were determined in trichloroacetic acid extracts of plasma and brain by paperchromatography and paperelectrophoresis. The plasma profiles indicated increased stability of the labile 8Lys-9Phe bond by the introduction of an 8D-Lys residue in the peptide analog. Enzymatic attack of the analog took place predominantly at 6His-7Phe and 7Phe-8D-Lys. Formation of tritiated water occcurred in brain and the gastro-intestinal tract and was considerable; proteolysis in these compartments was higher than in plasma. High uptake of radioactivity was found in the kidney, but urinary excretion was low during the first 30 min. Uptake in brain was low and paralleled uptake in cerebrospinal fluid. Intact peptide concentrations/g fresh tissue were in the order of 10(-5)-10(-4) times the administered dose for all three routes.