AbstractPituitary adenylate cyclase activating peptide (PACAP) is a member of the vasoactive intestinal peptide‐like peptide family. It is found in the hypothalamus, where the PACAP precursor is processed to form PACAP‐38, the C‐terminal truncated PACAP‐27 and PACAP related peptide (PRP). Both PACAPs are potent stimulators of anterior pituitary adenylate cyclase activity, but the physiologically relevant anatomical sources of PACAP and possible importance of PRP in this regard are poorly understood. Using immunocytochemistry with epitope‐specific antisera, we now show that PACAPS38, PACAP27‐ and PRP‐positive nerve fibres are all present in the rat median eminence. The major immunoreactive species present was PACAP38. Numerous PACAP38‐immmunoreactive nerve fibres were observed in the internal layer and a few were present in the posterior pituitary lobe. The external layer of the median eminence contained a few PACAP‐38‐immunoreactive fibres and PACAP‐38‐positive nerve terminals were rarely seen in the perivascular portal spaces. Surprisingly, delicate PACAP‐38‐positive nerve fibres were identified in the anterior pituitary lobe intermingled between the pituitary cells although none of the secretory pituitary cells contained immunoreactive PACAP38, PACAP27 or PRP and preproPACAP mRNA was not detected in the gland by Northern blotting or in situ hybridization. PACAP‐27‐ and PRP‐immunoreactive nerve fibres and terminals were found in the same locations as PACAP‐38 although generally in lower numbers. Specific radioimmunoassays and HPLC revealed that PACAP‐38 accounts for the vast majority of the adenohypophyseal PACAP‐immunoreactivity, whereas PACAP‐27 and PRP were found in low to undetectable concentrations. In primary cultures of rat pituitary cells and in the clonal gonadotrope‐derived aT3‐1 cell line, PACAP‐27 and PACAP‐38 were equipotent stimulators of cAMP accumulation, whereas PRP was ineffective. We conclude that the distribution of PACAP‐imrnunoreactive nerve fibres in the hypothalamus of the adult male rat is not that expected for a classic releasing factor suggesting that other sources of PACAP are relevant for stimulation of anterior pituitary cells or that the hypothalamic PACAP system is activated u