HPLC, gel chromatography, Northern analysis and non-isotopic in situ hybridization have been used to characterize for the first time the nature of vasoactive intestinal polypeptide mRNA and peptide in the rat thalamus and suprachiasmatic nucleus. Both these hitherto unstudied areas were shown to contain VIP-like immunoreactivity and VIP mRNA indistinguishable from those found in the rat cerebral cortex. Non-isotopic in situ hybridization with an alkaline phosphatase labelled antisense oligonucleotide specific for VIP mRNA and densitometry revealed that relative levels of VIP mRNA per cell were highest in the suprachiasmatic nucleus and lowest per cell in the thalamus of the rat brain areas investigated.