Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.