Targeting regulatable transgene expression specifically to neuronal or glial cell populations would facilitate studies of CNS gene function. We have developed the tetracycline (Tet) regulatable adenoviral system by expressing the Tet-off transactivator (tTA) under the control of the neuronal-specific synapsin I promoter and the well characterized glial-specific glial fibrillary acidic protein (GFAP) promoter. Transfection of primary hippocampal cultures demonstrated that the respective promoters restricted reporter transgene expression exclusively to neuronal or glial populations. Delivery of the vectors into adult rat hippocampus resulted in a similar pattern of cell specific transgene expression. These novel vectors provide a highly effective means of directing regulated, cell-specific, transgene expression and as such are important tools for investigations of neuronal and glial cell function and advancing gene therapy studies.