Antimutant activity of antimicrobials can be estimated by comparing drug pharmacokinetics with mutant prevention concentration (MPC). Large bacterial inocula known to reduce susceptibility have not been studied for effects on MPC determination.Staphylococcus aureus inoculum size was varied with solid and liquid media containing daptomycin and Ca(2+), a cation expected to lower inoculum effects, to assess effects on MIC and MPC.With drug-containing agar, individual colonies were obtained over a narrow range of inoculum size that shifted to higher inoculum size as daptomycin concentration increased. Increasing Ca(2+) supplementation from 1 to 50 mM lowered MIC by 2-fold and MPC from 20 to 3 mg/L, the latter determined by extrapolation of population analysis profiles to an inoculum size of 10(10) cfu. Cells of colonies recovered from daptomycin-containing agar had wild-type MIC. With liquid medium, supplemented with 1 mM Ca(2+)and containing 10(10) cfu, MPC was between 2.5 and 5 mg/L at an inoculum density of 10(7) cfu/mL. Bacteria recovered from liquid assays exhibited a 4- to 8-fold increase in MIC and contained point mutations in mprF.Inoculum effects on MPC can be reduced by measurement with low-density (large volume) liquid bacterial cultures. Retesting putative mutants for susceptibility can be important: stable mutants having genetic variations in the mprF gene were recovered from liquid medium, but not from agar. Daptomycin MPC with S. aureus was below minimal plasma drug concentration with approved doses, which is consistent with resistance to daptomycin arising rarely.