The principle hurdles for gene therapy are selectivity and efficacy. Toward that end, we constructed an adenovirus gene delivery system to enable robust, glial-specific, and repressible ectopic expression. A replication-incompetent (E1-deleted) adenovirus 5 vector was modified by the addition of three tandem repeats of a 300-bp fragment enhancer region of the glial fibrillary acidic protein gene coupled to a minimal promoter sequence from human cytomegalovirus to drive a tetracycline-controlled transactivator. Using beta-galactosidase as a reporter gene, we demonstrated high level expression in cells of glial origin (including cell lines derived from glioblastoma multiforme) but no detectable expression in nonglial cells (neuroblastoma or fibroblasts). Furthermore, expression was tightly regulated by anhydrous tetracycline. To our knowledge, this is the first gene therapy delivery system that is glial specific and which also allows for repression of ectopic gene expression.