The interactions of ceftobiprole with purified beta-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent beta-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 beta-lactamase, the class A TEM-1 beta-lactamase, and the class C AmpC beta-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum beta-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressing Enterobacter cloacae organisms. However, all three cephalosporins were hydrolyzed very slowly by AmpC cephalosporinases, suggesting that factors other than beta-lactamase stability contribute to lower ceftobiprole and cefepime MICs against many members of the family Enterobacteriaceae.