Chloramphenicol-resistant strains of Bacteroides fragilis (minimum inhibitory concentration, 12.5 mug/ml) were isolated from a stool specimen which contained multiply resistant Escherichia coli. The enzyme responsible for resistance, chloramphenicol acetyltransferase, was produced constitutively by these strains; the specific activity was 10-fold lower than that of the E. coli enzymes. Similar activity was not detected in susceptible B. fragilis strains, nor could it be induced by growth in the presence of chloramphenicol or by mutagenesis. The enzyme had a pH optimum of 7.8 and a molecular weight of approximately 89,000. The K(m) for chloramphenicol was 5.2 muM, and the enzyme was sensitive to inhibition by 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme produced by an E. coli strain isolated from the same specimen had a similar K(m) and sensitivity to 5,5'-dithiobis-2-nitrobenzoic acid.