The hair bundle, the hair cell's sensory organelle, transduces acoustical or vestibular stimulation into a change in membrane potential. The actin-based stereociliary processes of the hair bundle contain a number of myosin isoforms that may be important to the bundle's function. One of these isoforms, myosin Ibeta, has been proposed to constitute an adaptation motor controlling sensitivity of the hair bundle to mechanical displacement. To gain insight into myosin Ibeta's function, its distribution within the hair bundle was examined. A polyclonal antibody was produced that recognizes a protein surface loop within the head domain of myosin Ibeta. This antibody was used to localize myosin Ibeta in the hair cell by indirect-immunofluorescence microscopy and indirect-immunoelectron microscopy. Within the hair bundle, myosin Ibeta immunoreactivity was located along the sides of the stereociliary actin core, concentrated in the distal two-thirds of the stereocilia. Within the hair cell soma, myosin Ibeta immunoreactivity was located throughout the cytoplasm exclusive of the cuticular plate.