The selection of antibiotic-resistant mutant bacteria is proposed to occur in a drug concentration range (the mutant selection window) that extends from the minimum inhibitory concentration (MIC) of susceptible cells to the MIC of the least susceptible, single-step bacterial mutants (the mutant prevention concentration [MPC]). MPCs were estimated for tobramycin, chloramphenicol, rifampicin, penicillin, vancomycin, and several fluoroquinolones by use of Escherichia coli and Staphylococcus aureus. Comparisons among reported serum drug levels indicate that new fluoroquinolones are the least likely to enrich populations of resistant mutant bacteria during monotherapy. These data partly explain the selective enrichment of populations of resistant mutant bacteria in medical practice. The mutant selection window range (MPC:MIC) was narrowed for fluoroquinolones by structure modification, pointing to a new direction in antibiotic refinement. The mutant selection window and the MPC were determined for combinations of rifampicin and tobramycin, using S. aureus, as a guide for combination therapy with compounds that alone cannot block enrichment of mutant bacterial populations.