Glycine N-methyltransferase (GNMT) is the most abundant hepatic methyltransferase and plays important roles in regulating methyl group metabolism. In the central nervous system, GNMT expression is low and its function has not been revealed. The present study examines the effect of GNMT overexpression by adenovirus-mediated transfer in cortical mixed neuron-glial cultures. Infection of adenovirus encoding green fluorescence protein to cultures demonstrates high preference for non-neuronal cells. Optimal GNMT overexpression in cultures by adenoviral GNMT (Ad-GNMT) infection not only induces protein kinase C phosphorylation, but also increases neuronal/oligodendroglial survival. Furthermore, these Ad-GNMT-infected cultures are significantly resistant to H(2)O(2) toxicity and lipopolysaccharide stimulation. Conditioned media from Ad-GNMT-infected microglia also significantly enhance neuronal survival. Taken together, enhanced GNMT expression in mixed neuronal-glial cultures is neuroprotective, most likely mediated through non-neuronal cells.