Polymerase chain reaction (PCR) assay was compared with cell culture assay performed with use of HT29/C1 (human colonic epithelial) cells for identifying strains of enterotoxin-producing Bacteroides fragilis (ETBF) isolated from extraintestinal specimens. A total of 188 unselected strains obtained over 2 years at a central clinical laboratory in Tokyo were tested. Overall, 35 strains (18.6%) were positive by cell culture and PCR assay, 152 strains were negative by both assays, and 1 strain was negative by cell culture assay but positive by the PCR assay; the same results were obtained in repeated assays. Among 64 strains from blood, 18 (28.1%) were ETBF, a rate that was significantly higher (P < .05) than the 17 ETBF (13.7%) among 124 strains from other sites. These results suggest that PCR assay is a simple and reliable tool for detecting ETBF and that enterotoxin may be a virulence factor in bacteremia caused by B. fragilis.