ABSTRACT The efficacy of β-lactams is thought to be dependent on the time that the unbound concentrations exceed the MIC ( fT >MIC). However, the pharmacokinetic/pharmacodynamic index (PDI) that correlates best to the selection of resistance is not yet clear. The selection of ceftizoxime (CZX)-resistant Enterobacter cloacae mutant strains during the development of murine mixed-infection abscesses was studied to determine the PDI that is important for the emergence of resistance and the PDI value needed for the prevention of resistance. Studies were carried out 24 h after inoculation with Bacteroides fragilis ATCC 23745 and E. cloacae 22491. Six to 1,536 mg of CZX/kg of body weight/day given every 2 h (q2h), q4h, q6h, or q8h was started 30 min before inoculation and continued for 24 h. Resistant mutants were isolated to determine mutant frequencies (MF). The fT >MIC varied from 9 to 98% for E. cloacae , the peak concentration (unbound fraction) was 0.6 to 578 mg/liter, and the area under the concentration-time curve (unbound fraction) ( f AUC) was 1.9 to 553 mg·h/liter. The f AUC-to-MIC ratio best explained the in vivo efficacy. CZX-resistant B. fragilis and E. cloacae mutants were isolated from untreated controls at an MF of 10 ⁻⁵ to 10 ⁻⁷ . The MF of resistant B. fragilis did not increase during therapy. The selection of resistant E. cloacae strains at an MF of 10 ⁻¹ to 10 ⁻² was related to the fT >MIC and the ratio of f AUC to MIC following an inverse U shape. However, the ratio of f AUC to MIC was the stronger driver of resistance. The highest MFs were 0.7 to 0.9 at an f AUC-to-MIC ratio of approximately 250. We conclude that the ratio of f AUC to MIC is the PDI that correlated best to the in vivo efficacy of CZX and probably also to the emergence of resistant E. cloacae mutants. An f AUC-to-MIC ratio of 1,000 was needed to prevent the emergence of this resistance.