The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the A₁ agonist, 2-chloro-N⁶-cyclopentyladenosine (CCPA, 500 nM) or H₂O₂ (100 µM) for 15 minutes. Nuclear/myofilament, cytosolic, triton-soluble membrane and triton-insoluble membrane fractions were generated. CCPA and H₂O₂ activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In triton-soluble membrane fractions, H₂O₂ activated p38 MAPK and p42 ERK, while CCPA had no effect on MAPK activation in this fraction. The greatest difference between H₂O₂ and CCPA was in the triton-insoluble membrane fraction where H₂O₂ increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the triton-insoluble membrane fraction, suggesting that activation of this phosphatase may mediate CCPA effects in this fraction. The triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3 and greater than 85% of p38 MAPK and p42 ERK were bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 µM) reduced H₂O₂-mediated MAPK activation, but failed to attenuate CCPA-induced MAPK activation. H₂O₂ but not CCPA increased reactive oxygen species (ROS). Thus, adenosine A₁ receptor and oxidative stress differentially modulate subcellular MAPKs with the main site of divergence being the triton-insoluble membrane fraction. However, adenosine A₁ receptor-mediated MAPK activation does not involve ROS formation.