Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin bV, the mammalian b-heavy spectrin, as a myosin VIIa-and rhodopsin-interacting partner in photoreceptor cells. Spectrin bV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other b spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin bV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin bV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin bV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of a and b subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin bV can also form homodimers, which likely supports its aII-independent bV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin bV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin bV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin-and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.