Daptomycin resistance in Staphylococcus aureus emerged during therapy of tricuspid endocarditis. Susceptibility to daptomycin of the parent strain (SA-675), other daptomycin-susceptible strains and the non-susceptible mutant (SA-684) was heterogeneous; however, subpopulations growing at concentrations above the minimum inhibitory concentration (MIC) were not stably resistant. Stable resistance was produced only by serial passage on daptomycin-containing media. Daptomycin dissipated the membrane potential of SA-675 but not SA-684, which also lost an 81 kDa membrane protein. Whole cells and membranes of SA-684 bound a reduced amount of daptomycin. Reduced drug binding in SA-684 correlates with daptomycin resistance, possibly as a result of the loss of a membrane protein 'chaperone' with which daptomycin interacts. Heterogeneity of daptomycin MICs in susceptible strains may be an important factor in the development of stable, clinically relevant resistance.