The in vitro activity of daptomycin was studied in comparison with other agents active against 328 recent clinical isolates of Gram-positive pathogens. MIC data showed that the addition of calcium ions to a final concentration of 50 mg/L enhanced the activity of daptomycin generally by eight- to 16-fold. In the presence of calcium ions daptomycin was uniformly active against the strains of Staphylococcus spp. and Streptococcus spp. studied with a MIC90 of < or = 1 mg/L. Enterococcus faecalis and Enterococcus faecium were slightly less susceptible (MIC90 2 mg/L). Vancomycin-, fluoroquinolone- and quinupristin/dalfopristin-resistant strains were all susceptible to daptomycin. The presence of serum reduced the apparent activity of daptomycin to only a moderate extent. Employing the BSAC methodologies, a tentative breakpoint of 2 mg/L for daptomycin is proposed.