Microglial responses to endotoxin, including the synthesis of inflammatory factors, contribute to gliosis and neuron degeneration in cultured brain tissue. We have previously shown that Gö6976, a protein kinase C (PKC) inhibitor, suppressed the lipopolysaccharide (LPS)-induced production of inflammatory factors in microglia and afforded marked protection of neurons from glia-mediated cytotoxicity. The purpose of this study was to identify the signal transduction pathway underlying the neuroprotective effect of Gö6976. Gö6976 suppressed the LPS-induced release of tumor necrosis factor alpha (TNFalpha) in the microglial cell line, BV2. We show in this study the inhibitory effect of Gö6976 on TNFalpha release occurring through suppression of p38 mitogen-activated protein kinase (MAPK) phosphorylation and not through a PKC mechanism. While Gö6976 did not inhibit the activity of p38 MAPK directly, it did suppress its activation by phosphorylation, indicating the target of action of Gö6976 is a signaling event upstream of p38 MAPK. Although Gö6976 is considered a selective inhibitor of certain PKC isozymes, suppression of TNFalpha production was not mediated through inhibition of PKC activity. Gö6976 appears to play a novel role in neuroprotection by suppressing the release of pro-inflammatory factors by inhibiting the activation of p38 MAPK in microglia, rather than a PKC isoform.