The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinderplate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatographymass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific -lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum -lactamases and metallo--lactamases. Seven isolates of Bacteroides spp. produced multiple -lactamases, including possibly CepA, and metallo--lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading -lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that -lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple -lactamases from bacteria in the large intestines of cattle.