Previous studies in our laboratory have shown a differential activation of the mitogen-activated protein kinases (MAPKs) in primary bone marrow-derived macrophages following infection with pathogenic Mycobacterium avium compared to the activation following infection with nonpathogenic Mycobacterium smegmatis. Additionally, M. smegmatis-infected macrophages produced significantly elevated levels of tumor necrosis factor alpha (TNF-α) compared to the levels produced by M. avium-infected macrophages. The TNF-α production was dependent on both p38 and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. However, the macrophage transcription factors downstream of the MAPKs, which were required for TNF-α production, remained undefined. In this study we determined that the transcription factor cyclic AMP response element binding protein (CREB) is significantly more activated in M. smegmatis-infected macrophages than in M. avium-infected macrophages. We also found that CREB activation was dependent on p38 and protein kinase A but not on ERK 1/2 or calmodulin kinase II. Moreover, mutating the cAMP-responsive element on the TNF-α promoter resulted in significantly diminished promoter activity following M. smegmatis infection but not M. avium infection. The inability of macrophages infected with M. avium to sustain MAPK activation and to produce high levels of TNF-α was due, in part, to an increase in serine/threonine phosphatase PP2A activity. Our studies are the first to demonstrate an important role for the transcription factor CREB in TNF-α production by mycobacterium-infected macrophages, as well as a role for M. avium's induction of PP2A phosphatase activity as a mechanism to limit macrophage activation.