Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 microM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.