The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analyzed in middle and inner ear tissues from rats by use of microwave-aided fixation, a hyaluronan-binding protein and the avidin-biotin/peroxidase staining procedure. In the middle ear HYA was mainly localized in the pars flaccida, the ossicles, the round window membrane and in the endomysium of the middle ear muscles. The subepithelial stroma of the Eustachian tube was strongly HYA-positive. In the inner ear, the spiral ligament of the cochlea and the connective tissue surrounding the nerve fibers emerging from the crista ampullaris stained intensely for HYA. Except for a weak staining of the basilar membrane, the Corti organ was devoid of HYA. The heterogenous distribution of HYA may indicate its specific involvement in middle and inner ear physiology and pathology.