RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.