Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450(BM-1) in Bacillus megaterium has been provided in our previous report. In the present study, we show that the basal-level expression of the P450(BM-1) promoter-xylE transcriptional fusion is significantly reduced when the bm1P1 gene is present on the same plasmid. Moreover, disruption of the chromosomal bm1P1 gene results in enhanced expression of the P450(BM-1) promoter-xylE fusion on a multicopy plasmid. By using a binary vector system, we found that expression of the P450BM-1 promoter-xylE fusion from a high-copy plasmid is substantially repressed by expression of bm1P1 from a low-copy plasmid. Taken together, these results suggest that the basal-level expression of P450(BM-1) is negatively affected by bm1P1. Possible mechanisms for this control are discussed.