We have investigated the effects of two types of DNA-binding proteins on bacterial repression. First, the effects of operator positioning on repression by stationary DNA-binding proteins, the Lac repressor and the Trp repressor, were examined in vivo. Both operator number and positioning play a role in determining in vivo levels of repression. Operators located within a promoter are more efficient regulators than those positioned at the start of transcription. Second, we investigated the effects of DNA-binding protein density on repression using a mobile DNA-binding protein, Escherichia coli RNA polymerase. We employed a transcriptional interference assay using convergent transcriptional units. The strong synthetic promoter conI and its derivatives were observed to interfere with expression of the aadA gene, which confers spectinomycin resistance upon its host. Transcriptional interference by RNA polymerase occurred only in cis and had a strong dependence on polymerase density that was modulated by varying the promoter strengths. A change in the density of approximately fourfold completely abolished the observed transcriptional interference. Several models are discussed to explain the repression patterns observed for stationary and mobile DNA-binding proteins.