Lentiviral vectors offer well-recognized advantages as a gene delivery system both for the analysis of gene function and as a vehicle for gene therapy. In the present study optimized HIV-1-based vector systems that display efficient doxycycline (Dox)-dependent transgene expression in vitro and in vivo have been developed through the modification of factors that contribute to basal activity levels. Dissection of HIV-1 vectors harboring a tTA-dependent transgene expression cassette revealed several mechanisms that account for Dox-independent transgene expression, including those mediated by an internal CMV promoter, as well as a potential contribution from fusion proteins generated by translational readthrough. A precipitous reduction in basal activity levels was accomplished by separating the transactivator and the transgene cassettes into a binary vector system and by relocating the inducible promoter to the U3 region of the LTR. In addition, substituting the VP16 portion of tTA with the human p65 transactivating domain improved Dox-dependent transgene expression in a number of cell types. Optimizing HIV-1-based vectors culminated in a "toolbox" of vectors suitable for transgene delivery in vitro and in vivo, as conveyed by our ability to control the Dox-dependent differentiation of embryonic fibroblasts into muscle cells in vitro and transgene expression in rat brains.