In our previous publication (Shaw, G.-C., and Fulco, A. J. (1992) J. Biol. Chem. 267, 5515-5526), we reported that Bm3R1, a protein encoded in an open reading frame just upstream from the cytochrome P450BM-3 gene, is a repressor critically involved in the barbiturate-inducible expression of this gene in Bacillus megaterium. We now describe the purification of the Bm3R1 protein from an overproducing Escherichia coli strain harboring a bm3R1 gene-carrying plasmid and report the effect of barbiturate inducers on the interaction of Bm3R1 with a fragment of B. megaterium DNA containing the bicistronic operator and promoter sequences. Gel filtration analysis revealed that, under our experimental conditions, most of the Bm3R1 protein exists in highly aggregated forms. Gel mobility shift assays showed that Bm3R1 protein bound specifically to a segment of DNA containing the promoter-operator region of the bm3R1 gene while DNase I footprinting experiments established that Bm3R1 protected a region of DNA that covers and flanks the palindromic operator sequence. The interaction between Bm3R1 repressor and its operator, in vitro, was strongly inhibited by the addition of 2 mM pentobarbital or 2 mM methohexital (strong in vivo inducers of P450BM-3) but not by the same concentration of phenobarbital (a relatively weak inducer) or by mephobarbital (a non-inducer). A detailed comparison of pentobarbital and methohexital at concentrations lower than 2 mM indicated that methohexital was 5-10 times more effective as an inhibitor of Bm3R1 binding in vitro, as compared with its 7-fold greater inducer potency in vivo. The observation that the in vitro inhibition effects of barbiturates on the interaction of Bm3R1 repressor and its operator correlate strongly with their in vivo potency as inducers of cytochrome P450BM-3 suggests a mechanism for the induction process. It seems plausible that the barbiturate inducers might bear a conformational resemblance to and mimic the mode of action of an as yet unidentified endogenous inducer(s) in B. megaterium that functions by releasing the binding of Bm3R1 repressor from its operator site.