Different LPS mutants of Salmonella typhimurium and Salmonella minnesota have been investigated with respect to (1) their tendency to associate with HeLa cell monolayers, and (2) their physicochemical surface properties. Aqueous biphasic partitioning, hydrophobic interaction chromatography, and ion exchange chromatography have been used to characterize the bacterial cell surface properties with respect to charge and hydrophobicity. Liability to hydrophobic interaction was defined either by the change of partition in a dextran-polyethylene-glycol (PEG) system by the addition of PEG-palmitate (P-PEG), or by the elution pattern from Octyl-Sepharose. Accordingly, charge was asssessed by the effect of positively charged trimethylamino-PEG (TMA-PEG) on the partition, and by the elution from DEAE-Sephacel. Bacterial being negatively charged and liable to hydrophobic interaction had the highest tendency to associate with HeLa cells. In some cases the methods for surface analysis gave conflicting results on charge and/or liability to hydrophobic interaction of the same LPS mutant. Possible reasons for these differences and the role of bacterial cell surface structures contributing to physicochemical character are discussed.