The in vitro activity of daptomycin was assessed in comparison with that of vancomycin and penicillin against a wide range of Gram-positive aerobic clinical isolates. MICs were determined by an agar dilution method on Mueller-Hinton agar (NCCLS/EUCAST) and on Isotonic agar adjusted to contain 50 mg/L free calcium (BSAC). Both media were enriched with 5% horse blood for fastidious organisms. Daptomycin MICs for all 172 staphylococci, including methicillin-susceptible and methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus, were 0.03-0.5 mg/L. For 99 of the 100 enterococci (Enterococcus faecalis, n = 50; Enterococcus faecium, n = 50), including 37 vancomycin-resistant isolates, they were 0.25-2 mg/L. For all 108 beta-haemolytic streptococci, including Streptococcus pyogenes and Streptococcus agalactiae, daptomycin MICs were 0.016- 0.25 mg/L; for 101 alpha-haemolytic streptococci, including Streptococcus pneumoniae and 'viridans' streptococci, they were 0.016-2 mg/L. For miscellaneous vancomycin-resistant isolates including Lactobacillus spp., Lactococcus spp., Leuconostoc spp., Pediococcus spp. and isolates of Enterococcus casseliflavus and Enterococcus gallinarum, daptomycin MICs were 0.03-2 mg/L; MICs for the seven isolates of Listeria monocytogenes were 0.25-4 mg/L. There was little difference between the results on Mueller-Hinton agar and on supplemented Isotonic agar The discrepant results occasionally obtained tended to be one dilution higher on supplemented Isotonic agar. Daptomycin was active (MICs < or = 2 mg/L) against all the isolates tested with the exception of one isolate each of E. faecium and L. monocytogenes (MICs = 4 mg/L). Our results indicate that daptomycin MICs are independent of methicillin and vancomycin MICs.