We have characterized triple-helix-mediated inhibition of an artificial bacteriophage promoter with respect to relief of inhibition by incoming RNA polymerases that initiate upstream or downstream from the operator sequence. Whereas oligonucleotide-directed triple-helix formation inhibits the test promoter, promoter activity is restored when the triple-helical complexes are disrupted by transcription of either strand of the homopurine operator sequence. The degree of relief from inhibition is related to the frequency of operator transcription. These observations demonstrate that this artificial repressor-operator complex is subject to antagonism by cis elements (other promoters) acting at a distance. Such antagonism might also arise between certain natural transcriptional control regions. Our results suggest that the efficiency of artificial repressors based on triple-helix formation may be limited by transcriptional activity in the gene control region.