Methylmercury (MeHg), an organic methylated form of mercury, is one of the most hazardous environmental pollutants. MeHg is a potent neurotoxin, particularly during brain development. Neurotoxicity-induced by MeHg in prenatal age can cause mental disorders, cerebral palsy and seizures. We investigated cerebrospinal fluid (CSF) and brain tissue contents of S100B, a calcium binding protein produced and secreted by astrocytes, which has trophic and toxic activity on neurons depending on concentration. Pregnant rats were exposed to MeHg (5 mg/kg per day, on the 12th, 13th and 14th days of pregnancy). CSF and brain tissue (hippocampus, cerebral cortex and cerebellum) were obtained from neonate rats on 1, 15 and 30 days postnatal. MeHg accumulation was measured in brain tissue after birth and on the 30th postnatal day. An increase of CSF S100B was observed on the 15th, but not on the 30th postnatal day. Hippocampal tissue demonstrated increased S100B (and reduction in glial fibrillary acidic protein) immediately after birth, but not later. No changes in the S100B content were observed in cerebellum and cerebral cortex. No changes were observed in the spatial learning of these rats at adult age. These specific and reversible changes in the hippocampus could be related to the cognitive and epileptic disorders attributed to MeHg. Our results further indicate the glial involvement in the MeHg-induced neurotoxicity. The increment of CSF S100B in neonates exposed to MeHg reinforces the view that increased S100B is related to damage in the nervous system and that S100B could be a marker for MeHg-neurotoxicity. Although the cellular mechanism related to MeHg-induced increase in S100B content in CSF remains unknown, our results suggest the use of S100B as a peripheral marker of brain damage induced by MeHg.