pRevTet-On is a retroviral vector expressing the reverse tetracycline-controlled transactivator (rtTA) from the CMV promoter. This vector is derived from pLNCX, a retroviral vector created using elements of Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV) as described (1). rtTA is a fusion of amino acids 1–207 of the reverse tet repressor (rTetR) and the negatively charged C-terminal activation domain (130 amino acids) of the VP16 protein of Herpes Simplex Virus. rTetR was derived from TetR and differs by four point mutations, which are responsible for its opposite response to doxycycline. The 5' viral LTR controls expression of the transcript that contains Ψ+ (the extended viral packaging signal) and the neomycin resistance gene (Neor) for antibiotic selection in mammalian cells. rtTA is derived from vectors described previously (2–4). pRevTet-On also includes the E. coli Ampr gene for antibiotic selection in bacteria.