We have characterized the transcriptional regulation of ML-236B·Na and phenobarbital-inducible cytochrome P450 sca-2 (Cyt P450 sca-2) from Streptomyces carbophilus, an industrial pravastatin-producing strain. ML-236B·Na and phenobarbital enhanced the expression of the cytP450 sca-2 gene in S. carbophilus. The cytP450 sca-2 gene was also ML-236B·Na-inductive in S. lividans. Analysis of various deletion and mutation of the 5′-flanking region of the cytP450 sca-2 gene revealed that the 1-kb region was required for ML-236B·Na-dependent Cyt P450 sca-2 induction. We have found a putative ORF in the 5′-flanking region that encodes a protein of 174 amino acid residues containing a helix–turn–helix DNA-binding motif. A gel mobility shift assay showed that the protein was bound by an imperfect palindromic sequence between −46 bp and −24 bp in the 5′-flanking region, and ML-236B·Na was found to inhibit its binding. These findings suggest that induction of Cyt P450 sca-2 is negatively regulated at the transcriptional level and that the protein encoded by the putative ORF is possibly functional as a repressor of the cytP450 sca-2 gene.