As representative of gram-negative bacteria, the isolated and purified envelopes of an Escherichia coli K-12 strain were used to determine metal-binding capacity. The envelopes were suspended in 5 mM metal solutions for 10 min and 23 degrees C, separated and washed by centrifugation, and analyzed for metal by either atomic absorption or X-ray fluorescence spectroscopy. Of 32 metals tested, large amounts (> 0.9 mumol/mg [dry weight]) of Hf and Os, intermediate amounts (0.1 to 0.4 mumol/mg [dry weight]) of Pb, Zn, Zr, Fe III, Mn, Mo, Mg, Co, and Ce IV, and small amounts (< 0.1 mumol/mg [dry weight]) of Na, K, Rb, Ca, Sr, Cu, Sc, La, Pr, Sm, U, Fe II, Ru, Ni, Hg, Pt, Pd, Au, and In were detected Li and V were not bound to the envelopes. Electron microscopy of unstained, thin-sectioned material provided an electron-scattering profile for localizing the bound metal within the envelope. Energy-dispersive X-ray analysis of thin sections detected all metals in single envelope vesicles. These data suggest that most metal deposition occurred at the polar head group regions of the constituent membranes or along the peptidoglycan layer. No leaching of envelope components was detected by monitoring radioactive probes within the lipopolysaccharide and peptidoglycan layers during metal uptake experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from metal-loaded envelopes, or protein and carbohydrate determinations on the wash fluids. These results suggest that membrane integrity was not disturbed under these ionic conditions.