Adenosine inhibits growth of cardiac fibroblasts; however, the adenosine receptor subtype that mediates this antimitogenic effect remains undefined. Therefore, the goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat left ventricular cardiac fibroblasts, PDGF-BB (25 ng/mL) stimulated DNA synthesis (³H-thymidine incorporation), cellular proliferation (cell number), collagen synthesis (³H-proline incorporation), and MAP kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, but not N⁶-cyclopentyladenosine, 4-aminobenzyl-5′-N-methylcarboxamidoadenosine, or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A_2B receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A₂ receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A_2B receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5′-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A_2B receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of CF growth by activating A_2B receptors coupled to inhibition of MAP kinase activity. Thus, A_2B receptors may play a critical role in regulating cardiac remodeling associated with CF proliferation. Pharmacologic or molecular biological activation of A_2B receptors may prevent cardiac remodeling associated with hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.