Trimethyltin (TMT), is a hippocampal neurotoxicant characterized by neuronal degeneration, astrogliosis, and microglia reactivity with an associated elevation in proinflammatory cytokine mRNA levels. To examine the role of proinflammatory cytokines in the TMT-induced glia response, mixed cortical glia cultures were exposed to TMT and morphological and cytokine responses were examined. Morphological changes in the glia monolayer, enlarged, rounded cell bodies and retraction of the monolayer into distinct GFAP+ dense processes, displayed a dose (1, 5, and 10 μM TMT) and temporal response (6–48 h), accompanied by clustering of OX-42+ microglia. Tumor necrosis factor-α (TNF), interleukin (IL)-1α, and IL-6 mRNA levels were elevated by 3 and 6 h of TMT (10 μM) and proteins by 24 h. Recombinant proteins for IL-1α (100 pg/ml) and IL-6 (10 ng/ml) exacerbated the morphological response to TMT while those for TNFα (150 pg/ml) did not. Neutralizing antibodies (1:100) to IL-1α and IL-6 showed a slight decrease in the severity of the morphological response to TMT while, at 24 h, TNFα antibodies (1:100) and an antibody cocktail offered a significant level of protection. At 6 h, the neutralizing antibodies to TNFα or IL-1α did not elevate basal cytokine mRNA levels, however, IL-6 and the cocktail of antibodies significantly elevated IL-1α, IL-1β, and IL-6 mRNA levels. The specific elevation in IL-1α and IL-6 mRNA levels induced by TMT remained evident only in cells coexposed to anti-TNFα. Similar responses in cytokine mRNA levels were seen in cocultures of hippocampal neurons and glia exposed to TMT. These data suggest a relationship between microglia activation, proinflammatory cytokine release, and glia morphological responses, the significance of which remains to be determined, as well as, the impact on neuronal degeneration.