Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta -endotoxin genes. Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da. The data revealed that the delta -endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta -endotoxin gene. DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region. The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta -endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans. The results showed that Japanese isolates of B. thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta -endotoxin allied to the class Cry9. It also appeared that heterogeneous multiple H serovars are involved in each group.