The organophosphorus (OP) pesticide malathion is a highly neurotoxic compound. Although some studies have reported neurotoxicity signs after the in utero exposure to OP pesticides, there is no evidence of the exclusive contribution of the lactational exposure to malathion as a possible cause of neurotoxicity in the offspring. In this study, we investigated the exclusive contribution of malathion exposure through maternal milk on the activity of acetylcholinesterase (AChE), as well as on biochemical parameters related to the oxidative stress (glutathione levels, lipid peroxidation and glutathione reductase and glutathione peroxidase activities) in the brain of suckling mice. The same parameters were also evaluated in the brains of the respective mothers, which where directly exposed to malathion during the lactational period (daily s.c. injections; doses of 20, 60 and 200mg/kg of body weight). Our results showed that the lactational exposure to malathion caused a high inhibitory effect of the brain AChE activity in the offspring, even when dams were exposed to the lowest malathion dose (20mg/kg). Brain AChE activity was also inhibited in mothers; however, only at the highest malathion dose (200mg/kg). No changes were observed in the biochemical parameters related to the oxidative stress for both dams and pups brains. The present study shows, for the first time, that the exposure of neonatal mice to malathion via lactation inhibits the activity of brain AChE in the offspring. These data, summed to the fact that OP pesticides are excreted in human milk, makes relevant the lactational exposure to these xenobiotics in terms of human health concerns.