Abstract Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two molecular forms: PACAP-38 and PACAP-27. Soon after the isolation and chemical characterization of PACAP, the first radioimmunoassay (RIA) methods have been developed, but it is a still rarely used laboratory technique in the field of PACAP research. The aim of the present study was to develop a novel, highly specific PACAP-38 assay to investigate the quantitative distribution of PACAP-38 in the central nervous system of various vertebrate species under the same technical and experimental conditions. Different areas of the brain and the spinal cord were removed from rats, chickens and fishes and the tissue samples were processed for PACAP-38 RIA. Our results indicate that the antiserum used in the RIA is C-terminal specific, without affinity for other members of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon peptide family. The average ID 50 value was 48.6±3.4 fmol/ml determined in 10 consecutive assays. Detection limit for PACAP-38 proved to be 2 fmol/ml. PACAP-38 immunoreactivity was present in the examined brain areas of each species studied, with highest concentration in the rat diencephalons. High levels of PACAP-38 were also detected in the rat telencephalon, followed by spinal cord and brainstem. The central nervous system of the fish also contained considerable concentrations of PACAP-38, whereas lowest concentrations were measured in the central nervous system of the chicken.