Unique strains of Bacillus thuringiensis, that belong to the four H serogroups (serovar sumiyoshiensis, serovar fukuokaensis, serovar darmstadiensis, and serovar japonensis) and produce spherical parasporal inclusions specifically toxic to lepidopteran larvae, were examined for comparative analysis of the genes encoding delta-endotoxin proteins. Gene analysis revealed that there is no difference between the four strains in nucleotide sequences of the 1,937-bp DNA segment covering the four conserved regions and a partial sequence of the block 5 region. Surprisingly, the nucleotide sequence of the four strains showed a 100% homology with that of the corresponding region of the cry9D gene encoding a delta-endotoxin protein, which had been reported to be active on the scarabaeid coleopterans. Ali,Alignment analysis revealed that the N-terminal half (16-660) amino acid sequence of the four proteins shared relatively high homologies (27.7-35.8%) with those of the Cry9Ba, Cry9Ca, and Cry1Ba proteins, while lower homologies with those of the Cry3Aa, Cry8Ca, and Cry1Aa proteins. The results show that the cry9D gene is retained in multiple heterogeneous ii serovars of Lepidoptera-specific B. thuringiensis populations naturally occurring in soil environments of Japan.