Interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha are both thought to be critical factors in the defence against mycobacteria but are known to play different roles. In this study, we investigated the regulatory pathways for IL-12 and TNF-alpha expression in human monocyte-derived macrophages (MDMs) after treatment with Mycobacterium tuberculosis H37Rv or the Triton X-100 solubilized proteins (TSP) purified from M. tuberculosis. We found a rapid phosphorylation of Akt and extracellular signal-regulated kinase (ERK), albeit with differential activation kinetics, in human MDMs treated with M. tuberculosis or TSP. Studies using inhibitors selective for phosphatidylinositol 3-kinase (PI 3-K) and ERK 1/2 show that both pathway plays an essential role in the induction of TNF-alpha at both the transcriptional and translational levels in human MDMs. In contrast, blockade of the PI 3-K/Akt or ERK 1/2 pathways significantly increased M. tuberculosis- or TSP-induced IL-12 p40 and p35 mRNA and bioactive p70 protein. The enhancement of IL-12 levels by inhibition of PI 3-K and ERK 1/2 was not reversed by neutralization of TNF-alpha or addition of rhTNF-alpha, suggesting that the negative regulation of IL-12 is not mediated by concomitant TNF-alpha suppression. Further, PI 3-K activity is required for the M. tuberculosis- or TSP-induced phosphorylation of ERK 1/2 activation. TSP from M. tuberculosis shows a similar dependency on the PI 3-K and ERK 1/2 pathways to those by M. tuberculosis. Collectively, these data suggest that the Th1-driving cytokine IL-12 and proinflammatory cytokine TNF-alpha are differentially regulated by PI 3-K and ERK 1/2 pathways in human MDMs during mycobacterial infection. These results may provide therapeutic targets for precise and specific fine-tuning of cytokine responses.