Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multidrug resistant infections, including those caused by methicillin-resistant S. aureus strains. Similar to many other antimicrobial peptides, daptomycin binds with preference to anionic membranes such as those typically found in prokaryotes. However, in contrast to most linear a-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions, and its activity in vivo is absolutely Ca2+-dependent. Here, we describe the early events that occur in the binding of daptomycin to lipid bilayers using a quantitative model to analyze both equilibrium and kinetic binding data. The goal of the analysis was to obtain a precise description of the early events that occur in the interaction of daptomycin with lipid and calcium ions at low daptomycin concentrations. In the course of the analysis, we also determined the rate and equilibrium constants for binding of daptomycin to lipid and Ca2+. The model used to describe the experimental data comprises a soluble daptomycin monomer that binds calcium ions in solution with low affinity, a soluble, Ca2+-bound dimer, and a 1:1 daptomycin-lipid(Ca) complex. A strong interaction of daptomycin with Ca2+-complexed lipid, the amount of which depends on the availability of calcium ions in the bulk solution, appears central to its function.