Solid-state NMR has been used to examine the binding of N'-4-[(4-fluorophenyl)benzyl)]chloroeremomycin, a fluorinated analogue of oritavancin, to isolated protoplast membranes and whole-cell sucrose-stabilized protoplasts of Staphylococcus aureus, grown in media containing [1(13)C]glycine and L-[epsilon-(15)N]lysine. Rotational-echo double-resonance NMR was used to characterize the binding by estimating internuclear distances from (19)F of oritavancin to (13)C and (15)N labels of the membrane-associated peptidoglycan and to the (31)P of the phospholipid bilayer of the membrane. In isolated protoplast membranes, both with and without 1 M sucrose added to the buffer, the nascent peptidoglycan was extended away from the membrane surface and the oritavancin hydrophobic side chain was buried deep in the exposed lipid bilayer. However, there was no N'-4-[(4-fluorophenyl)benzyl)]chloroeremomycin binding to intact sucrose-stabilized protoplasts, even though the drug bound normally to the cell walls of whole cells of S. aureus in the presence of 1 M sucrose. As shown by the proximity of peptidoglycan-bridge (13)C labels to phosphate (31)P, the nascent peptidoglycan of the intact protoplasts was confined to the membrane surface.