The G_i-coupled adenine receptor (AdeR), binds adenine with high affinity, and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex (CTX) and outer medulla (OM) as compared to inner medulla (IM). Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid C-terminal sequence of rat AdeR which we generated, detected two bands between ~30-40 kDa (MW of native protein 37 kDa) in CTX, OM and IM. These bands were ablated by pre-adsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature including the glomerular arterioles, and less intense labeling in the cells of the collecting duct (CD) system. Confocal immunofluorescence imaging co-localized AdeR with AQP2 protein to the apical plasma membrane in the CD. Functionally, adenine (10 µM) significantly decreased (P < 0.01) dDAVP (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 µM; P < 0.01)), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in renal vasculature and collecting ducts, and its functional relevance. This study may open a new avenue for exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease.