S100b, a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100b. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100b in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM) or vehicle for 1day (acute) or 7 days (chronic). RT-PCR and ELISA determined S100b mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100b in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05). Acute and chronic lithium treatments exerted no significant effects on intracellular S100b protein levels in astrocytes, and extracellular S100b protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100b mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100b protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100b level, with no effect on astrocytes or interaction between neurons and astrocytes.