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A heterologous RIA method for pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a homologous RIA method for a shorter form of PACAP with 27 residues (PACAP27) were established to determine PACAP content in central and peripheral tissues in rats. The highest concentration of radioimmunoassayable PACAP38 was found in the hypothalamus, but other brain regions also contained considerable amounts of PACAP38. PACAP38 concentration in the posterior pituitary was comparable with that in the extrahypothalamic brain, but its concentration in the anterior pituitary was very low. Unexpectedly, the testis contained a high abundance of PACAP38, and the total amount of PACAP in both testes exceeded its content in the whole brain. Reverse phase HPLC suggested that the major testicular PACAP38 immunoreactivity represents PACAP38. Among peripheral tissues, adrenal gland contained the second highest concentration of PACAP. Smaller amounts of PACAP were widely distributed in the digestive tract and other peripheral tissues. The concentration of PACAP in stomach, duodenum and jejunum appeared to be greater than in other portions of the gut. In all tissues, PACAP27 represented only a minor portion of total PACAP immunoreactivity.