AbstractMultiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK‐3/6, MKK‐1/2 and MKK‐4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP‐1. Exposure of N9 microglia to Mn (250 µm), LPS (100 ng ml−1) or Mn + LPS increased MKK‐3/6 and MKK‐4 activity at 1 h; the effect of Mn + LPS on MKK‐4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK‐3/6 and MKK‐1/2 phosphorylation, whereas MKK‐4 was activated only by Mn and Mn + LPS. Besides activating MKK‐4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK‐4's phosphorylation on Ser80, which negatively regulates MKK‐4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP‐1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF‐α, IL‐6 and Cox‐2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK‐1/2, MKK‐3/6 and MKK‐4, are activated by Mn suggests that Mn‐induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn‐induced activation was persistent at least for 4 h, indicating a long‐term effect. Copyright © 2010 John Wiley & Sons, Ltd.