Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesion for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer), gangliotetraosylceramide (Gal beta 1-3 GalNAcT beta 1-4 Gal beta 1-4Glc beta 1-1Cer), and lactosylceramide (Gal beta 1-4Glc beta 1-1Cer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesion of P. aeruginosa.