In an effort to develop a regulatable derivative of the promoter of the human gene for U6 snRNA, we generated several constructs composed of the human U6 snRNA promoter and sequences derived from the gene for the tetracycline operator of a prokaryotic tetracycline resistance transposon. One of the constructs had strong transcriptional activity in the presence of tetracycline that was equivalent to 80% of the activity of the wild-type promoter. Furthermore, the transcriptional activity was almost completely repressed in the absence of tetracycline. Transcriptional activity became detectable within 4 hr after the addition of tetracycline to the culture medium. We used this system to control the functional activity of an antisense RNA for a chimeric gene derived from genes for the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP). A plasmid that expressed the chimeric gene and a plasmid that expressed the antisense RNA under the control of the inducible U6 promoter were used to cotransfect HeLa cells that were producing the tetracycline repressor protein (Tet R). Addition of tetracycline to the culture medium 12 hr after transfection resulted in the almost complete disappearance of the fluorescent signal due to the chimeric protein within 24 hr. Our results suggest that this expression system might be a useful tool for controlling the expression of functional RNAs, such as aptamers and antisense RNAs, both in basic research and in gene therapy.