Strains of Bacteroides fragilis that produce an extracellular 20-kD heat-labile toxin have been epidemiologically associated with diarrheal disease. To standardize detection of the B. fragilis toxin (BFT), a detailed description of the methods for identifying BFT production by B. fragilis strains is reported. To further study the role of toxigenic strains of B. fragilis in clinical disease, extraintestinal clinical isolates that were recovered at an urban east-cost hospital were evaluated. Four (6.2%) of 65 isolates produced BFT. Three cases in which BFT-producing B. fragilis were isolated from extraintestinal sites are reported. In contrast to studies from Japan that have reported BFT production by 23.4% of extraintestinal B. fragilis isolates, we conclude that BFT-producing strains of B. fragilis are infrequently associated with extraintestinal B. fragilis disease at our institution. Additional study will be necessary to better define the importance of BFT production in human disease due to B. fragilis.