The role of renal A₁ adenosine receptors (AR) in the pathogenesis of radiocontrast nephropathy is controversial. We aimed to further elucidate the role of A₁AR in the pathogenesis of radiocontrast nephropathy and determine whether renal proximal tubule A₁AR contribute to the radiocontrast nephropathy. To induce radiocontrast nephropathy, A₁AR wild type (WT) or knockout (KO) mice were injected with a non-ionic radiocontrast (iohexol, 1.5-3 grams iodine/kg). Some A₁WT mice were pretreated with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, a selective A₁AR antagonist) before iohexol injection. A₁AR contribute to the pathogenesis of radiocontrast nephropathy in vivo as the A₁WT mice developed significantly worse acute renal failure, more renal cortex vacuolization and had lower survival 24 hrs after iohexol treatment compared to the A₁KO mice. DPCPX pretreatment also protected the A₁WT mice against radiocontrast induced acute renal failure. No differences in renal cortical apoptosis or inflammation were observed between A₁WT and A₁KO mice. To determine whether the proximal tubular A₁AR mediate the direct renal cytotoxicity of radiocontrast, we treated proximal tubules in culture with iohexol with or without 2-chloro-N⁶-cyclopentyladenosine (CCPA, a selective A₁AR agonist) or DPCPX pretreatment. We also subjected cultured proximal tubule cells over-expressing A₁AR or lacking A₁AR to radiocontrast injury. Iohexol caused direct dose-dependent reduction in proximal tubule cell viability as well as proliferation. Neither the A₁AR agonist nor the antagonist treatment affected proximal tubule viability or proliferation. Moreover, over-expression or lack of A₁AR failed to impact the iohexol toxicity on proximal tubule cells. Therefore, we conclude that radiocontrast causes acute renal failure via mechanisms dependent on A₁AR, however, renal proximal tubule A₁AR do not contribute to the direct tubular toxicity of radiocontrast.