Abstract Colistin-susceptible isolates of Acinetobacter baumannii often contain subpopulations that are resistant to colistin.Monotherapywithcolistincanleadtoselectivegrowth of these subpopulations and emergence of colistin-resistant strains.Ourobjectivesweretoexplorethesusceptibilitypattern of colistin-resistant subpopulations and investigate if combiningcolistinwithasecondantibioticcouldpreventtheirselective growth. Four colistin-susceptible clinical isolates of A. baumannii and one reference isolate were used. The mutant prevention concentration (MPC) of colistin, i.e. the concentration required to block growth of all single step-mutant subpopulations,wasdeterminedbyplatinganinoculumof109CFUon Mueller Hinton agar (MHA) plates containing 2-fold dilutions of colistin (0.125–128 mg/L). Susceptibility testing of colistinresistant subpopulations, obtained in the MPC assay, was performed with Etest. The MPC of colistin, in combination with rifampicin,wasdeterminedbyplatinganinoculumof109 CFU onMHA platescontainingcolistin(0.125–128mg/L)andfixed concentrations of rifampicin (1.1 mg/L or 4.4 mg/L). The colistin-resistant subpopulations demonstrated increased susceptibility to a number of agents compared to their main populations. These subpopulations were even susceptible to agents thatnormallyareinactiveagainstgram-negativebacteriaandall had rifampicin MICs of