Cephalosporinase activity was demonstrated in all of 10 strains of Bacteroides fragilis investigated. Low rates of hydrolysis of cephalosporins (0.25 to 3.5 μmol of cephaloridine per h per 10 9 cells) were found, but no activity against penicillin substrates was detected. In two strains the cephalosporinase activity was increased 40-and 80-fold by growing cells in the presence of penicillin. No permeability barrier for these antibiotics was demonstrated. In most cases the substrate profile showed decreasing activity in the following order: cephaloridine > cephalothin > cephaloglycin > cephalexin. The cephalosporinase from these organisms differed from that found in facultative and aerobic gram-negative bacilli in that it was inhibited by both cloxacillin and p -chloromercuribenzoate. Among nine strains of Bacteroides fragilis subspecies fragilis , correlation was found between in vitro resistance to cephaloridine and amount of β-lactamase activity in sonically disrupted cells.