Energy depletion increases the renal production of 2',3'-cAMP (a positional isomer of 3',5'-cAMP that opens mitochondrial permeability transition pores) and 2',3'-cAMP is converted to 2'-AMP and 3'-AMP, which in turn are metabolized to adenosine. Because the enzymes involved in this "2',3'-cAMP-adenosine pathway" are unknown, we examined whether 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) participates in the renal metabolism of 2',3'-cAMP. Western blotting and real time PCR demonstrated expression of CNPase in rat glomerular mesangial, preglomerular vascular smooth muscle and endothelial, proximal tubular, thick ascending limb and collecting duct cells. Real time PCR established the expression of CNPase in human glomerular mesangial, proximal tubular and vascular smooth muscle cells; and the level of expression of CNPase was greater than that for phosphodiesterase 4 (major enzyme for the metabolism of 3',5'-cAMP). Over-expression of CNPase in rat preglomerular vascular smooth muscle cells increased the metabolism of exogenous 2',3'-cAMP to 2'-AMP. Infusions of 2',3'-cAMP into isolated CNPase wildtype (+/+) kidneys increased renal venous 2'-AMP, and this response was diminished by 63% in CNPase knockout (-/-) kidneys; whereas the conversion of 3',5'-cAMP to 5'-AMP was similar in CNPase +/+ versus -/- kidneys. In CNPase +/+ kidneys, energy depletion (metabolic poisons) increased kidney tissue levels of adenosine and its metabolites (inosine, hypoxanthine, xanthine and uric acid) without accumulation of 2',3'-cAMP. In contrast, in CNPase -/- kidneys, energy depletion increased kidney tissue levels of 2',3'-cAMP and abolished the increase in adenosine and its metabolites. Conclusion: Kidneys express CNPase, and renal CNPase mediates in part the renal 2',3'-cAMP-adenosine pathway. Key Words: 2',3'-cyclic adenosine monophosphate; 2'-adenosine monophosphate; 3'-adenosine monophosphate; adenosine; 2',3'-cyclic nucleotide 3'-phosphodiesterase; CNPase