Unconventional myosins have been associated with hearing loss in humans, mice, and zebrafish. Mutations in myosin VI cause both recessive and dominant forms of nonsyndromic deafness in humans and deafness in Snell's waltzer mice associated with abnormal fusion of hair cell stereocilia. Although myosin VI has been implicated in diverse cellular processes such as vesicle trafficking and epithelial morphogenesis, the role of this protein in the sensory hair cells remains unclear. To investigate the function of myosin VI in zebrafish, we cloned and examined the expression pattern of myosin VI, which is duplicated in the zebrafish genome. One duplicate, myo6a, is expressed in a ubiquitous pattern during early development and at later stages, and is highly expressed in the brain, gut, and kidney. myo6b, on the other hand, is predominantly expressed in the sensory epithelium of the ear and lateral line at all developmental stages examined. Both molecules have different splice variants expressed in these tissues. Using a candidate gene approach, we show that myo6b is satellite, a gene responsible for auditory/vestibular defects in zebrafish larvae. Examination of hair cells in satellite mutants revealed that stereociliary bundles are irregular and disorganized. At the ultrastructural level, we observed that the apical surface of satellite mutant hair cells abnormally protrudes above the epithelium and the membrane near the base of the stereocilia is raised. At later stages, stereocilia fused together. We conclude that zebrafish myo6b is required for maintaining the integrity of the apical surface of hair cells, suggesting a conserved role for myosin VI in regulation of actin-based interactions with the plasma membrane.