Abstract The accumulation of reactive microglia in the degenerating areas of amyotrophic lateral sclerosis (ALS) tissue is a key cellular event creating a chronic inflammatory environment that results in motoneuron death. We have developed a new culture system that consists in rat spinal cord embryonic explants in which motoneurons migrate outside the explant, growing as a monolayer in the presence of glial cells. The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) have been proposed to be involved in ALS-linked microglial activation. In our explants, the combined exposure to these cytokines resulted in an increased expression of the pro-oxidative enzymes inducible nitric oxide synthase (iNOS), the catalytic subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91 phox and cyclooxygenase-2 (COX-2), as compared to each cytokine alone. This effect was related to their cooperation in the activation of the transcription factor nuclear factor kappa B (NF-κB). TNF-α and IFN-γ also cooperated to promote protein oxidation and nitration, thus increasing the percentage of motoneurons immunoreactive for nitrotyrosine. Apoptotic motoneuron death, measured through annexin V-Cy3 and active caspase-3 immunoreactivities, was also found cooperatively induced by TNF-α and IFN-γ. Interestingly, these cytokines did not affect the viability of purified spinal cord motoneurons in the absence of glial cells. It is proposed that the proinflammatory cytokines TNF-α and IFN-γ have cooperative/complementary roles in inflammation-induced motoneuron death.