Previous reports have documented that a surface layer protein (SlpA) varies among Clostridium difficile isolates. The typing system by sequencing the variable region of the slpA gene was applied to typing C. difficile strains belonging to one PCR ribotype, type smz, which has been identified as frequently causing outbreaks in Japan. The PCR ribotype smz strains recovered from patients at different hospitals in Japan were examined. Among 10 type smz strains tested, three subtypes, smz-1, -2 and -3, were identified that differed from each other by one nucleotide. slpA sequence typing was also applied to direct typing on DNA extracted from stool specimens. Of 22 stool specimens examined, 17 were PCR positive for slpA; eight were typed as slpA sequence type smz-1 and nine as type smz-2. C. difficile was cultured from 12 of these 17 stool specimens, and the sequence results of the recovered isolates were compared with those from the DNA extracted from the stool specimens. In all 12 of these stool specimens, the sequence results of DNA from recovered C. difficile isolates completely agreed with those of DNA extracted directly from stool specimens. The remaining five stool specimens were culture-negative for C. difficile. Sequence typing has the advantage of enabling easy comparison of typing results among multiple laboratories via the Internet without exchanging reference strains as is required in typing systems which depend on banding-pattern analyses. slpA sequence typing appears to be a reproducible and reliable typing system for C. difficile as well as being useful for the typing of C. difficile when stool specimens contain only small numbers of C. difficile or are inappropriate for culturing.