To understand how cells differentially use the dozens of myosin isozymes present 
in each genome, we examined the distribution of four unconventional myosin 
isozymes in the inner ear, a tissue that is particularly reliant on actin-rich 
structures and unconventional myosin isozymes. Of the four isozymes, each from a 
different class, three are expressed in the hair cells of amphibia and mammals. 
In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is 
found mostly near stereociliary tips, myosin-VI is largely absent, and 
myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In 
the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, 
myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These 
three myosin isozymes are excluded from other actin-rich domains, including the 
circumferential actin belt and the cortical actin network. A member of a fourth 
class, myosin-V, is not expressed in hair cells but is present at high levels in 
afferent nerve cells that innervate hair cells. Substantial amounts of 
myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is 
largely free of F-actin, squeezed between (but not associated with) actin of the 
cuticular plate and the circumferential belt. Our localization results suggest 
specific functions for three hair-cell myosin isozymes. As suggested previously, 
myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in 
cuticular plates and association with stereociliary rootlets suggest that this 
isozyme participates in rigidly anchoring stereocilia; and finally, 
colocalization with cross-links between adjacent stereocilia indicates that 
myosin-VIIa is required for the structural integrity of hair bundles.