Polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate or sodium deoxycholate (SDS- or DOC-slab-PAGE) is a powerful technique for the separation of smooth(S)-type bacterial lipopolysaccharides (LPS). In order to recover the individual LPS species from the polyacrylamide gel for subsequent analyses, a sensitive, nondestructive reverse staining of slab-PAGE-separated LPS has been developed. The individual reverse-stained LPS bands can be rapidly and efficiently recovered into an aqueous 5% triethylamine solution when they are extruded to produce fine gel microparticles. Based on these principles, an isolation methodology that combines preparative slab-PAGE, reverse staining, extrusion, and passive elution can be used to isolate, to electrophoretic homogeneity, micrograms to hundreds of micrograms of individual LPS species successfully from smooth-type LPS mixtures.