Adenosine is elaborated in injured tissues where it suppresses inflammatory responses of essentially all immune cells, including production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the A(2A) adenosine receptor (A(2A)AR). Previously, however, it has been shown that the A(3)AR agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA) potently inhibited TNF-alpha release from macrophages obtained from A(2A)AR "knockout" (A(2A)KO) mice, suggesting that the A(3)AR may also regulate cytokine expression. Here, we confirmed that the A(2A)AR is the primary AR subtype that suppresses TNF-alpha release from thioglycollate-elicited mouse peritoneal macrophages induced by both Toll-like receptor-dependent (TLR) and TLR-independent stimuli, but we determined that the A(2B)AR rather than the A(3)AR mediates the non-A(2A)AR actions of adenosine since 1) the ability of IB-MECA to inhibit TNF-alpha release was not altered in macrophages isolated from A(3)KO mice, and 2) the A(2B)AR antagonist 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine (MRS 1754) blocked the ability of the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA) to inhibit TNF-alpha release from macrophages isolated from A(2A)KO mice. Although A(2B)ARs seem capable of inhibiting TNF-alpha release, the A(2A)AR plays a dominant suppressive role since MRS 1754 did not block the ability of NECA to inhibit TNF-alpha release from macrophages isolated from wild-type (WT) mice. Furthermore, the potency and efficacy of adenosine to inhibit TNF-alpha release from WT macrophages were not influenced by blocking A(2B)ARs with MRS 1754. The data indicate that adenosine suppresses TNF-alpha release from macrophages primarily via A(2A)ARs, although the A(2B)AR seems to play an underlying inhibitory role that may contribute to the anti-inflammatory actions of adenosine under select circumstances.