The internal transcribed spacers between the 16S and the 23S ribosomal RNA genes were used to discriminate species of the 16S rRNA group I of the genus Bacillus by PCR. The spacer-PCR fingerprints clearly discriminated the different species, except those closely related like the members of the 'B. cereus group' (B. cereus, B. thuringiensis and B. mycoides) and the species of the 'B. subtilis group' (B. amyloliquefaciens and B. licheniformis). Examining in more detail the shortest internal transcribed spacers, B. subtilis group species were distinguished by single-strand conformation polymorphism analysis, whereas B. mycoides was differentiated from B. cereus/B. thuringiensis by restriction analysis.