The Etest manufacturer (bioMerieux) recommends Mueller-Hinton agar (MHA) with calcium concentration of 25-40 mg/L for daptomycin testing. A two phase study was performed to evaluate the effect of European agars on daptomycin Etest MICs. Broth microdilution (BMD) testing was performed and compared to Etest MICs for a challenge set of 20 Staphylococcus aureus with daptomycin MICs near the susceptible breakpoint and S. aureus 29213. In the first phase, Etest MICs were determined using agar plates prepared from MHA, IsoSensitest Agar (ISA) and Brain-Heart Infusion Agar (BHIA) and supplemented with various levels of calcium. In the second phase, Etest MICs were determined using commercially prepared MHA from Mast (MST), BD, Oxoid (OX), BioRad (BR), bioMerieux (BM) and E&O (EO) and ISA plates from OX, EO and MST. The calcium concentration of each agar was determined using ion selective electrode methodology. The best correlation of Etest and BMD MICs was obtained in phase 1 using MHA with 42.8 mg/L calcium and in phase 2 with BD MHA. The phase 2 MHA calcium concentration ranged from 19.2 to 63.6 mg/L. 87.5% of strains with BMD MICs of 1 mg/L, had Etest MICs of 1.5 or 2 mg/L using MHA with recommended calcium levels. Etest MICs using BHIA and ISA were higher than the BMD MICs and therefore are not recommended for use with the daptomycin Etest. The high percentage of major errors using Etest in this study, even with use of the most optimal medium, suggests that Etest MICs of 1.5 or 2 mg/L using MHA should be retested by BMD.