Daptomycin MICs were determined for 844 Gram-positive bacteria in three concentrations of Ca(++) and compared with the MICs of vancomycin and teicoplanin. Daptomycin was twofold to fourfold more active against most species when tested in 50 microg/ml of Ca(++) than in 25 microg/ml. In 50 microg/ml of Ca(++) daptomycin was more active against methicillin-resistant staphylococci and vancomycin-resistant enterococci than teicoplanin or vancomycin; 100% of these isolates were susceptible to < or =2.0 microg/ml of daptomycin. Different lots of Mueller-Hinton agar were variable in Ca(++) content, and daptomycin disk diffusion zone diameters were affected, i.e., zones were 1 to 15 mm smaller on one lot of agar with only 6 microg/ml of Ca(++) compared to another lot with 28 microg/ml. The previously proposed daptomycin interpretive breakpoints performed satisfactorily when MICs were determined in Mueller-Hinton broth with 50 microg/ml of Ca(++) and when the agar gave appropriate zones with quality control strains. To define those control limits, replicate tests with four quality control strains were performed in ten laboratories using broth microdilution tests (with Ca(++) supplemented broth) and disk diffusion tests on Mueller-Hinton agar without cation adjustments.