We have used C{F}, N{F}, and N{P} rotational-echo double resonance NMR to determine the location and conformation of F-19 and N-15 double-labeled plusbacin A(3) and of double-labeled deslipo-plusbacin A3, each bound to the cell walls of whole cells of Staphyloccocus aureus grown in media containing [1-C-13]glycine. The P-31 is primarily in wall teichoic acid. Approximately 25% of plusbacin headgroups (the cyclic depsipeptide backbone) are in a closed conformation (N-F separation of 6 angstrom), while 75% are in a more open conformation (N-F separation of 12 angstrom). The closed headgroups have no contact with wall teichoic acid, whereas the open headgroups have a strong contact. This places the closed headgroups in hydrophobic regions of the cell wall and the open headgroups in hydrophilic regions. None of the plusbacin tails have contact with the P-31 of either wall teichoic acid or the cell membrane and thus are in hydrophobic regions of the cell wall. In addition, both heads and tails of plusbacin A(3) have contact with the glycyl C-13 incorporated in cell-wall peptidoglycan pentaglycyl bridges and with C-13-labeled purines near the membrane surface. We interpret these results in terms of a dual mode of action for plusbacin A(3): first, disruption of the peptidoglycan layer nearest to the membrane surface by closed-conformation plusbacin A(3) leading to an inhibition of chain extension by transglycosylation; second, thinning and disruption of the membrane (possibly including disruption of ATP-binding cassette transporters embedded in the membrane) by open-conformation plusbacin A(3), thereby leading to release of ATP to the hydrophilic regions of the cell wall and subsequent binding by plusbacin A(3).