The purpose of this work was to determine the effect of melatonin on the nitric oxide levels in murine splenocytes cultured with the Venezuelan equine encephalomyelitis virus. After incubation, nitric oxide levels were measured by the diazotization assay. Those cultures with the Venezuelan equine encephalomyelitis virus increased nitric oxide levels. Splenocytes infected and treated with 100 and 150 microg/ml of melatonin, decreased significantly the nitric oxide levels when compared to infected and non-treated splenocytes. These findings show that splenocytes infected with the Venezuelan equine encephalomyelitis virus generate important amounts of nitric oxide and suggest that melatonin protects the mice infected with the Venezuelan equine encephalomyelitis virus by a mechanism involving the decreasing of nitric oxide concentrations in tissue.