The cryIVB gene of Bacillus thuringiensis subsp. israelensis (Bti) codes for a 135-kDa insecticidal crystal protein, which is specifically toxic to dipteran larvae. We have identified a transcription start point (tsp) of cryIVB by a primer extension experiment. The promoter sequence alignment, together with the chronology of appearance of the transcript, suggested that cryIVB is transcribed by RNA polymerase containing sigma 35 (E sigma 35). This was confirmed by investigation of cryIVB transcription in several Bacillus subtilis sporulation mutants. Unlike the lepidopteran-specific crystal protein-encoding genes [cryIA(a) and cryIB], transcription of which is regulated by both sigma 35 and sigma 28, cryIVB transcription was controlled only by the sigma 35-dependent promoter at the midsporulation stage.