The role of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of TNF-⍺ and NOS2 production by human monocytes infected with Mycobacterium bovis BCG was examined. Inhibition studies showed that ERK1/2 and p38 MAPK activation were necessary for the monocyte response to M. bovis infection. Analysis of MAPK activation showed rapid phosphorylation of ERK1/2 and p38 in response to M. bovis BCG. Phosphorylation was not due to an autocrine effect of TNF-⍺ secretion, since an anti-TNF-⍺ antibody had no significant effect on the levels of p38 phosphorylation. The inhibitor PD98059 significantly reduced M. bovis BCG-induced TNF-⍺ production and almost completely abrogated phosphorylation of ERK1/2; in addition the potent MEK inhibitor U0126 also abrogated phosphorylation. In contrast, studies using inhibitors selective for ERK1/2 and p38 showed that p38 plays an essential role in the induction of NOS2, whereas the role of ERK1/2 was minor. These results suggest that ERK1/2 and p38 kinases differentially regulate the M. bovis BCG-mediated induction of TNF-⍺ and NOS2 in human monocytes.