The primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been determined and the results show that the sequence of PACAP has been highly conserved during evolution. In particular, the structure of the 1-27 fragment of PACAP is identical in frog and mammals. Using an antiserum raised against PACAP27, we have investigated the distribution of PACAP-containing neurons in the central nervous system of the frog Rana ridibunda by the immunofluorescence technique. The main populations of immunoreactive perikarya were located in the medial and ventral diencephalon, i.e., the preoptic nucleus, the ventral and dorsal infundibular nuclei, the nucleus posterocentralis thalami, and the ventral and ventrolateral areas of the thalamus. In the telencephalon, sparse cell bodies were found in the nucleus accumbens septi, the amygdala, the pallial commissure, and the bed nucleus of the pallial commissure. In the hindbrain, the torus semicircularis, the nucleus profundus and the nucleus anteroventralis tegmenti of the mesencephalon also contained populations of PACAP-immunoreactive perikarya. Beaded nerve fibers were observed throughout the brain. Occasionally they formed bundles, e.g., from the ventral infundibulum to the external vascular layer of the median eminence, from the central thalamus to the optic tectum, and rostrocaudally, from the nucleus accumbens septi to the nucleus entopeduncularis. Other areas, such as the interpeduncular nucleus, the nucleus isthmi and the roots of cranial nerves V and VIII in the medulla oblongata, were also densely innervated. The adenylate cyclase-stimulating activity of PACAP was tested by using a static incubation technique for hypothalamic slices.(ABSTRACT TRUNCATED AT 250 WORDS)