Using high-speed scanning atomic force microscopy, we directly observed single-molecular enzymatic elongation of hyaluronan polymer chains at intervals of 10 s on a mica or lipid bilayer surface, on which Pasteurella multocida hyaluronic acid synthase (pmHAS) was immobilized. The reaction was started by the addition of both UDP-glucuronic acid and UDP-N-(a)cetylglucosamine monomers. The average catalytic elongation rate constant (k(cat)) was found to be 1.8 mer s-I from one active enzyme physically adsorbed on a mica surface. When pmH.AS was immobilized by inserting its hydrophobic tail part into lipid bilayers, most of the enzymes retained their activity, and the k, values were found to be in the range 1-10 mer s(-1) for 29 enzymes (average was k(cat) = 2-4 mer s(-1)). These c, values were lowest level of k(cat) = 1-100 s(-1) obtained in bulk solution by radioisotope methods.