Staphylococcus aureus, coagulase-negative staphylococci, and Enterococcus spp. represent the most frequently recovered organisms from bloodstream infections. As a treatment option, daptomycin has been recently approved for the treatment of S. aureus bacteremia and right-sided endocarditis. We evaluated the spectrum of activity and potency of daptomycin and other antibiotics against 142 linezolid-nonsusceptible clinical strains. The isolates were tested for susceptibility by reference broth microdilution methods utilizing physiologic free calcium ions levels (50 mg/L) when testing daptomycin. Staphylococcus spp. and Enterococcus spp. were selected and screened for 23S rRNA, L4 and L22 mutations, and the cfr gene. Daptomycin was potent against all linezolid-nonsusceptible staphylococci (minimal inhibitory concentrations [MIC](90), 0.5 microg/ml) and enterococci (MIC(90), 1 microg/ml) isolates at the respective breakpoints of <or=1 microg/ml and <or=4 microg/ml. The majority of the isolates (84.5%) showed ribosomal target-site alterations, mainly G2576T, and five isolates (two S. aureus and three Staphylococcus epidermidis) had the mobile cfr element. In conclusion, daptomycin was the most active agent tested against this collection of gram-positive clinical organisms and ribosomal target mutations comprised the main resistance mechanism against linezolid.