Vasoactive intestinal peptide (VIP) has been proposed as an inducer of PRL release. Moreover, immunocytochemical studies suggested an increase in the VIP content in the hypothalamus of lactating rats. We investigated whether this increase is regulated at the level of gene transcription. A sensitive RNA detection assay that uses in vitro transcribed RNA probes corresponding to specific exons of the VIP gene was devised to study VIP gene expression. Using this method, an approximately 2000-base long RNA containing the coding sequences for VIP was detected in rat hypothalamus. This RNA also contains the coding sequences for the VIP-related peptide PHM/I (peptide histidine methionine amide or isoleucine amide). An identical VIP-encoding RNA was previously identified in the rat cerebral cortex. A 2-fold increase was observed in hypothalamic VIP mRNA during lactation. In addition, the levels of this mRNA increased in the hypothalamus at the time of sexual maturation. Taken together, our results suggest a physiological regulation of VIP gene expression associated with its potential role as a neuroendocrine hormone.