Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D-and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor.