A sensitive hapten-sandwich immunofluorescence technique was used to examine binding of lipopolysaccharide (LPS) at the single-cell level. The structural features governing such binding to murine lymphocytes were investigated by evaluating LPS binding in the presence of a variety of charged molecules and after different target-cell treatments. Polymyxin B, the positively charged proteins egg-white lysozyme and protamine chloride, and the polyanion dextran sulfate inhibited LPS binding to murine lymphocytes. Pretreatment with the proteolytic enzyme pronase and the cross-linking agent paraformaldehyde abolished the capacity of lymphocytes to bind LPS. Inhibition by polymyxin B was less effective when added 30 min after initiation of incubation of LPS with cells at 0 C or when added at the initiation of incubation at 37 C. These results suggest that the interaction between LPS and lymphocytes is a two-stage process, the first of which is dependent on a positively charged membrane protein. The second stage is postulated to be an irreversible hydrophobic interaction between LPS and membrane lipids.