Adenosine is released into the extracellular space from nerve terminals and cells subjected to ischemic stress. This nucleoside modulates a plethora of cellular functions via occupancy of specific receptors. Adenosine is also an important endogenous regulator of macrophage function, as it suppresses the production of a number of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-12, macrophage inflammatory protein (MIP)-1α and increases the release of the anti-inflammatory cytokine IL-10 by these cells. However, the mechanisms of these anti-inflammatory effects have not been well characterized. We hypothesized that adenosine may exert some of its anti-inflammatory effects by decreasing activation of the transcription factor nuclear factor κB (NF-κB), because gene expression of most of the proinflammatory cytokines inhibited by adenosine is dependent on NF-κB activation. Using LPS-stimulated RAW 264.7 macrophages, we found that adenosine as well as adenosine receptor agonists decreased the production of TNF-α, a typical NF-κB-regulated cytokine. This effect of adenosine was not due to an action on the process of TNF-α release, as adenosine suppressed also the intracellular levels of TNF-α. However, cDNA microarray analysis revealed that mRNA levels of neither TNF-α nor other cytokines were altered by adenosine in either LPS-activated or quiescent macrophages. Furthermore, adenosine as well as adenosine receptor agonists failed to decrease LPS-induced NF-κB DNA binding, NF-κB promoter activity, p65 nuclear translocation and inhibitory κB (IκB) degradation. We also hypothesized that adenosine would activate cyclic adenosine 5’-monophosphate (cAMP) response element binding protein (CREB) in macrophages since adenosine receptor stimulation has been associated with activation of the cAMP–protein kinase A system as well as of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, all of which can activate the CREB transcription factor system. We found that extracellular adenosine enhanced CREB transcriptional activity and increased phosphorylation of nuclear CREB. On the other hand, adenosine failed to alter CREB DNA binding. Adenosine stimulated both p38 and p42/44 MAPK activation. The p38 MAPK pathway inhibitor SB203580 but not the p42/44 MAPK pathway blocker PD98059 decreased adenosine-induced CREB activation indicating that p38 MAPK but not p42/44 MAPK is an upstream mediator of CREB activation. Thus, some of the immunomodulatory effects of adenosine in macrophages may be explained by its augmenting effect on CREB activation. Taken together, our results suggest that the anti-inflammatory effects of adenosine are independent of NF-κB but may be related by its augmenting effect on CREB activation.