CD73 metabolizes extracellular 5′-AMP to adenosine; yet recent experiments in brain tissue suggest that CD73 is not required for the metabolism of 5′-AMP to adenosine because of tissue nonspecific alkaline phosphatase (TNAP), which like CD73 is a GPI-anchored ecto-enyzme with 5′-nucleotidase activity. Because adenosine importantly regulates renovascular function, we investigated whether both TNAP and CD73 are involved in the renovascular metabolism of 5′-AMP. To test this, we examined in isolated, perfused mouse kidneys the metabolism of 5′-AMP (applied to the lumen of the renal vasculature via intrarenal artery administration) to adenosine by measuring renal venous levels of 5′-AMP, adenosine, and inosine (adenosine metabolite) by mass spectrometry. In one study, we compared 5′-AMP metabolism in naive CD73+/+ (wild-type, n = 16) vs. CD73−/− (knockout, n = 16) kidneys; and in a second study, we compared 5′-AMP metabolism in CD73+/+ ( n = 9) vs. CD73−/− ( n = 8) kidneys pretreated with levamisole (1 mmol/l; TNAP inhibitor). In naive kidneys, 5′-AMP increased renal venous 5′-AMP, adenosine, and inosine, and these responses were similar in CD73+/+ vs. CD73−/− kidneys. Levamisole per se did not inhibit renovascular 5′-AMP metabolism; however, in the presence of levamisole, 5′-AMP increased renal venous 5′-AMP threefold more in CD73−/− vs. CD73+/+ kidneys and knockout of CD73 inhibited 5′-induced adenosine and inosine by 81 and 86%, respectively. TNAP mRNA, protein, and activity were similar in CD73+/+ vs. CD73−/− kidneys. In conclusion, CD73 and TNAP play interactive roles to metabolize luminally applied 5′-AMP in the renal vasculature such that inhibition of both is required to inhibit the production of adenosine.