We have recently demonstrated protection against renal ischemic-reperfusion injury in vivo by A₁- and A_2a-adenosine receptor (AR) modulations. To further elucidate the signaling cascades of AR-induced cytoprotection against reperfusion/oxidant-mediated injury, immortalized human proximal tubule (HK-2) cells were treated with H₂O₂. H₂O₂ caused dose- and time-dependent HK-2 cell death that was measured by lactate dehydrogenase release and trypan blue dye uptake. Adenosine protected against H₂O₂-induced HK-2 cell death by means of A₁- and A_2a-AR activation. A₁-AR-mediated protection involves pertussis toxin-sensitive G proteins and protein kinase C, whereas A_2a-AR-mediated protection involves protein kinase A activation by means of cAMP and activation of the cAMP response element binding protein. Moreover, protein kinase A activators (forskolin and Sp-isomer cAMP) also protected HK-2 cells against H₂O₂ injury. De novo gene transcription and protein synthesis are required for both A₁- and A_2a-AR-mediated cytoprotection as actinomycin D and cycloheximide, respectively, blocked cytoprotection. Chronic treatments with a nonselective AR agonist abolished the protection by adenosine. Moreover, chronic treatments with a nonselective AR antagonist increased the endogenous tolerance of HK-2 cells against H₂O₂. We concluded that A₁- and A_2a-AR activation protects HK-2 cells against H₂O₂-induced injury by means of distinct signaling pathways that require new gene transcription and new protein synthesis.