␤-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce ␤-exotoxin I have suggested that there is a strong correlation between high levels of ␤-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry ؊)(Pig ؉), with no crystal gene, produced considerable amounts of ␤-exotoxin I together with a soluble brown melanin pigment. Therefore, ␤-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of ␤-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry ؊)(Pig ؉) mutants. We screened for nonpigmented mutants with impaired ␤-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for ␤-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for ␤-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers ␤-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule.