Optimizing pharmacokinetic/pharmacodynamic indices of antibiotics to obtain clinical and microbiological efficacy is essential, but dosing regimens must also be tailored to minimize the risk for emergence of resistance. The aim of the present study was to investigate whether certain concentrations of benzylpenicillin are critical for the selection of resistant subpopulations. A mixed culture of Streptococcus pneumoniae containing ca. 90% susceptible (MIC = 0.031 mg/liter), 9% intermediate (MIC = 0.25 mg/liter), and 1% resistant (MIC = 8 mg/liter) was studied in an in vitro kinetic model. The time that concentrations exceeded the MIC (T>MIC) for the three strains in the culture was varied by different initial concentrations of benzylpenicillin. Samples for viable counts were withdrawn at different times during 24 h and seeded on blood agar plates and on selective antibiotic-containing plates. The T>MIC varied from 46 to 100% for the susceptible strain, from 6 to 100% for the intermediate strain, and from 0 to 48% for the resistant strain. Our study, which may mimic the clinical situation with carriage of a mixed population of S. pneumoniae with different antibiotic susceptibilities, has shown that selection of resistant bacteria may easily occur if dosing regimens are only targeted toward fully susceptible strains.