This study characterized the upstream signalling molecules involved in extracellular signal-regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)-alpha expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF-alpha expression in human primary monocytes and monocyte-derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB-induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) zeta decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF-alpha induction caused by MTBRv or MTBRa. Toll-like receptor (TLR) 2 was found to play a major role in MTB-induced TNF-alpha expression and PKCzeta phosphorylation. Co-immunoprecipitation experiments showed that PKCzeta interacts physically with TLR2 after MTB stimulation. Moreover, PKCzeta phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCzeta interacts with TLR2 to play an essential role in MTB-induced ERK 1/2 activation and subsequent TNF-alpha expression in monocytes/macrophages.