The minimum inhibitory concentrations (MIC) of phosphates (0.1% sodium ultraphosphate, UP; 0.1% sodium polyphosphate glassy, SPG; 0.5% sodium acid pyrophosphate, SAPP; 0.5% sodium tripolyphosphate, STPP; and 0.5% tetrasodium pyrophosphate, TSPP) were bactericidal to early exponential phase cells of Staphylococcus aureus ISP40 8325 in a pH 6 modified complete defined synthetic medium. Concentration effects were observed for each phosphate in which the higher the concentration, the greater the inhibition. Chain-length effects were observed for those phosphates in which the longer the chain-length, the greater the inhibition. Leakage of intracellular nucleotides were confirmed spectrophotometrically (release of A[subscript]260-absorbing material) and microscopically (appearance of gelatinous cellular aggregates). The bactericidal and bacteriolytic effects of phosphates indicated that the cell envelope was damaged. The target site of phosphates was defined at the cell wall. The bactericidal effects of UP (0.1%) and SPG (0.1%) were reversed by Ca[superscript]2+ (0.01 M) or Mg[superscript]2+ (0.01 M) through metal-rescue when either Ca[superscript]2+ or Mg[superscript]2+ was added to rescue cells one hour after UP or SPG addition. No additive effects existed between Mg[superscript]2+ and Ca[superscript]2+ in the metal-rescue study. In contrast, the growth inhibition of TSPP was reversed by Fe[superscript]3+ by metal-protection because Fe[superscript]3+ protected cells when added one hour before the addition of TSPP. Results supported the hypothesis that long-chain polyphosphates (UP and SPG) interacted with S. aureus cell walls by a metal-ion chelation mechanism. In addition, long-chain polyphosphates were shown to bind to the cell wall, chelate metals, and remain bound, without releasing the metals into the culture medium;A hypothesis is proposed to explain the antibacterial mechanism of long-chain polyphosphates. The long-chain polyphosphates bind to the cell walls of early exponential phase cells of S. aureus ISP40 8325 and chelate structurally essential metals (Ca[superscript]2+ and Mg[superscript]2+) of the cell walls resulting in cell death and lysis. The structurally essential metals are probably those that form cross bridges between the teichoic acid chains in the cell walls of Gram-positive bacteria.