Daptomycin (LY146032) caused a calcium-dependent dissipation of the membrane potential (delta psi) in Staphylococcus aureus without noticeably affecting the chemical gradient (delta pH) across the membrane. The effect of daptomycin on membrane energization may account for many of the inhibitory effects on macromolecular biosyntheses and membrane function reported for this antibiotic. Our evidence indicates that the bactericidal activity of daptomycin is dependent on an available delta psi.