—Adenosine inhibits growth of vascular smooth muscle cells. 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 aortic vascular smooth muscle cells, platelet-derived growth factor–BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([³H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([³H]proline incorporation), total protein synthesis ([³H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5′-N-methylcarboxamidoadenosine, but not N⁶-cyclopentyladenosine 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 MAP kinase activity. 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 vascular smooth muscle cell growth by activating A_2B receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A_2B receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis following balloon angioplasty.