Adenoviruses are highly efficient vectors for gene transfer into brain cells. Restricting transgene expression to specific cell types and maintaining long-term expression are major goals for gene therapy in the central nervous system. We targeted gene expression to neurons by constructing an adenoviral vector that expressed the E. coli LacZ reporter gene under the control of the rat neuron-specific enolase promoter (Ad-NSE). Expression from Ad-NSE was compared with that from an adenoviral vector encoding the same reporter gene under the control of the Rous sarcoma virus LTR promoter (Ad-RSV). Both recombinant adenoviruses were injected stereotactically into rat hippocampus, cerebellum and striatum. Anatomical and immunohistochemical analyses of the Ad-NSE-stained cells showed that neurons were preferentially transduced. More neurons were stained in the hippocampus following infection with Ad-NSE than with Ad-RSV. Cytotoxicity from Ad-NSE was lower than from Ad-RSV. beta-Galactosidase gene expression after Ad-NSE infection remained stable for 3(1/2) months, and was detectable for 6 months. Thus, the NSE-adenoviral vector can be used to transfer potentially therapeutic genes into neuronal cells. The use of a cell-specific promoter also resulted in high in vivo efficiency and long-term transgene expression.