Bacillus thuringiensis Cry8Ea toxin is specifically toxic to larvae of the Asian cockchafer, Holotrichia parallela. Here we investigated the mechanism of transcriptional regulation of the cry8Ea1 gene. Reverse transcription-PCR (RT-PCR) results indicated that cry8Ea1 and an upstream gene (orf1) were cotranscribed. Transcriptional fusions with the lacZ gene demonstrated that transcription of the cry8Ea1 gene started from two promoters: P(orf1), which is located upstream of the orf1 gene, and P(cry8E), located in the intergenic region mapping between orf1 and cry8Ea1. Of the known, similar orf1-cry operons, this is the first report of the existence of a promoter in the intergenic region between the orf1 and cry genes. The transcriptional activity of P(orf1) was found during sporulation in B. thuringiensis subsp. kurstaki HD-73 and was almost abolished in the sigE mutant, while the transcriptional activity of P(cry8E) was detected after the end of the exponential phase in HD-73 and was considerably lower in the sigH mutant. The transcription start sites generated by the two cry8Ea1 promoters were determined by the 5' -SMARTer rapid amplification of cDNA ends (RACE) method. The -35 and -10 regions of P(orf1) and P(cry8E) showed high sequence similarity with the σ(E) and σ(H) promoters, respectively. These results indicated that P(orf1) is controlled by the σ(E) factor and P(cry8E) by the σ(H) factor.