Recombinant adeno-associated virus (rAAV) vector supports long-term transgene expression from skeletal muscle in most mammals, including human. In some instances, the requirement for tight control of the transgene expression is expected. The original tetracycline-dependent system using the rtTA (Dox-on) transactivator displayed a baseline activity in the off state but improved versions are now available and need to be evaluated in a single-rAAV-vector strategy. In the present study we cloned, in three different orientations, the two expression cassettes responsible for doxycycline-mediated transgene regulation and further evaluated the basal and inducible activity of the recently described rtTA2(S)-S2, rtTA2(S)-M2, and rtTA2(S)-M2n/s transactivators. Evaluations were conducted in vivo in mice and nonhuman primates using the respective homologous erythropoietin cDNA as a reporter gene because of its sensitive detection by ELISA. The woodchuck hepatitis virus posttranscriptional regulatory element sequence was also introduced to enhance further the stringency with respect to basal activity in the absence of inducer.