The concentration of hyaluronan (hyaluronic acid; sodium hyaluronate; HA), a major component of connective tissue, was analyzed by a specific radioassay in tissue samples from normal rat middle ears and in tissue and effusions from middle ears affected by experimentally induced otitis media. In normal ears the pars flaccida of the tympanic membrane contained a considerably higher concentration of HA (60 to 200 micrograms/g) than the pars tensa and areas of the medial wall (2 to 7 micrograms/g). In purulent otitis media the pars flaccida contained less HA than in normal ears. The HA concentrations were lower in purulent (3 to 40 micrograms/mL) than in serous (20 to 190 micrograms/mL) effusions. The weight average molecular weight of HA in serous fluid was high (greater than 10(6] and comparable to that in lymph. It is inferred from the study that the subepithelial tissue and its matrix components, eg, HA, must be considered when attempting to understand the function of the middle ear normally and in otitis media.