Adenosine A_2B receptors have been suggested to influence cell differentiation and proliferation. Human adenosine A_2B receptors expressed in Chinese hamster ovary cells mediate phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2). Already low concentrations of agonists such as 5′-N-ethylcarboxamidoadenosine (NECA) are effective. Phosphorylation of the stress-activated protein kinase p38 was also potently induced by NECA (EC₅₀ 18.5 nM). These NECA-induced effects were mimicked by forskolin and 8-Br-cAMP. Inhibition of cAMP-dependent protein kinase (PKA) using H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide)) blocked phosphorylation of the cAMP response element-binding protein (CREB) and p38, but did not decrease NECA-induced ERK1/2 phosphorylation. NECA activated the small GTPase Rap1, and this was also not blocked by H89. Inhibition of phosphatidylinositol-3′-kinase (PI3K) by wortmannin inhibited adenosine A_2B receptor-mediated ERK1/2 phosphorylation and activation of Rap1, without affecting CREB and p38 phosphorylation. A_2B receptor-stimulated protein kinase B phosphorylation was sensitive to wortmannin, but not to H89. Thus, stimulation of adenosine A_2B receptors activates both ERK1/2 and p38 via cAMP, but the downstream pathways are markedly different. ERK1/2 activation was dependent on PI3K but not on PKA. p38 activation by NECA was instead independent of PI3K but required cAMP and PKA. The potent activation of both MAPKs suggests a physiological role.