Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that greatly stimulates adenylyl cyclase activity in cultured anterior pituitary cells, was isolated from ovine hypothalamus in 1989. Investigation of the distribution of PACAP messenger RNA (mRNA) in rat tissues by Northern blot analysis revealed an anomalous signal in the testis. In this study we have isolated and characterized this unusual mRNA, which is approximately 800 bases long (approximately 1.5 kilobases shorter than that reported in the rat hypothalamus). Cloning and sequencing of the complementary DNA corresponding to this message revealed that the sequences are identical except for 126 bases at the 5'-end of the 5'-untranslated region of the smaller transcript. This region has no homology to either the published hypothalamic sequence or any other known sequence. Northern blot analysis of total RNA from various species showed that a smaller form of PACAP mRNA is also present in human, murine, and bovine testis, although in these species the message is slightly smaller. In addition, Northern blot analysis of these tissues using a probe directed to the 126-base 5'-region, revealed conservation of this sequence between species. Although the structure of the rat PACAP gene is unknown, preliminary investigations into the origins of the two mRNA species by PCR of genomic DNA suggests that they are transcribed from separate genes and not the product of alternate splicing.