A single lentivirus vector allowing doxycycline-regulated expression of transgenes in the brain was generated by incorporating the tetracycline (Tet)-dependent regulatory system into the backbone of the vector. Two distinct expression cassettes were inserted upstream and downstream from the central Flap sequence that provides for enhanced transduction of nondividing cells. The first cassette was used to express the transgene under the control of the Tet-dependent minimal cytomegalovirus promoter. The second cassette was employed to express constitutively the Tet-dependent transactivator rtTA2-M2, which activates the Tet-dependent promoter after binding of doxycycline (Tet-on system). Vectors carrying luciferase and tyrosine hydroxylase as the transgene were constructed, tested in astroglia-rich primary cultures, and injected into the striata of rats. The constructs allowed in vitro and in vivo robust expression of the transgene that could be regulated over two orders of magnitude by the addition and withdrawal of doxycycline. The vector may thus be useful for many applications in gene therapy research, including the development of a therapeutic protocol for the treatment of Parkinson's disease based on the restoration of regulated dopamine production.