Daptomycin is highly active against Staphylococcus aureus, including multidrug-resistant strains and those with reduced susceptibility to vancomycin. However, daptomycin-non-susceptible (Dap(NS)) strains [minimum inhibitory concentration (MIC) >1mg/L] have been derived clinically and in vitro. The mechanism(s) by which this occurs is incompletely understood, but existing data indicate that it is multifactorial. Dap(NS) derivatives of one laboratory and three clinical strains of S. aureus produced using gradient plates were evaluated. The Dap(NS) phenotype included increases in glycopeptide and nisin MICs and in some instances defective autolysis and reduced susceptibility to lysostaphin lysis. Amino acid substitutions in MprF, YycG (WalK), or both, were identified in all Dap(NS) strains. Reduced cytochrome c binding and ability of daptomycin to depolarise whole cells correlated with the Dap(NS) phenotype, consistent with an increase in cell surface positivity. Gene expression data revealed increased expression of vraS, one member of a two-component system involved in the regulation of cell wall biosynthesis, in three of five Dap(NS) strains. The pathway to the Dap(NS) phenotype is not linear, as variable genetic and phenotypic changes may result in identical increases in MICs.