Adenosine is an endogenously released autocoid that has potent receptor-mediated modulatory effects on macrophage function. The intracellular pathways mediating these effects are incompletely understood. Since adenosine receptor occupancy has been associated with activation of the cAMP-PKA system as well as of p38 MAPK and p42/44 MAPK, all of which can activate the CREB transcription factor system, we hypothesized that adenosine would activate CREB in macrophages. Using RAW 264.7 macrophages, 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.