Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without Loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a Laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a Laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.