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The effect of melatonin (MLT) on the brain levels of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in Venezuelan equine encephalomyelitis (VEE) virus infection was determined. Brain homogenates from mice inoculated with 10 LD50 of VEE virus, untreated or treated with 500μg MLT/kg body weight were assayed by ELISA to measure the levels of TNF-α and IL-1β. MLT was injected daily starting 3 days before and continuing to 7 days after virus inoculation. Infected mice treated with MLT showed decreased levels of TNF-α when compared to the untreated infected mice on days 1, 3, 4, and 5 postinoculation (P < 0.001). In contrast, IL-1β levels increased from days 1 to 5 in the infected mice treated with MLT when compared with the untreated infected animals (P < 0.01). The results suggest that the protective effect of MLT on the VEE virus infection could be due, among other factors, to a decrease in TNF-α synthesis along with an increase in the production of IL-1β.