The intermediate filament (IF) proteins paranemin and synemin are unique members of the IF protein superfamily. Paranemin and synemin were originally identified as IF-associated proteins because they colocalize and copurify with desmin in avian muscle cells. The IFs wrap around the periphery of myofibrils at their Z-lines in adult skeletal muscle cells, connect adjacent myofibrils to one another and to any nearby mitochondria and nuclei, and connect the peripheral layer of myofibrils to the sarcolemma at costameres. We have followed the colocalization of paranemin, synemin, and desmin during skeletal muscle myogenesis at different stages of development by immunofluorescence and immunoelectron microscopy. Localization of paranemin precedes that of desmin in cultured avian skeletal muscle presumptive myoblasts. Paranemin, synemin and desmin then colocalize throughout muscle cell development but, in relation to desmin and synemin, paranemin immunofluorescence is increased at the growth tips of elongating myotubes and decreased in areas of the cell where the myofibrils are in a more mature pattern of alignment. The IF proteins colocalized within longitudinal filaments dispersed around/between myofibrils, but there was no IF colocalization with the myofibrillar Z-line protein α-actinin until after myofibril alignment had occurred. At the ultrastructural level, IFs were observed near the ends of early nascent myofibrils and at cytoskeletal filament junctions. A direct interaction of paranemin with both desmin and synemin was demonstrated by in vitro binding assays. Furthermore, expression of GFP-tagged paranemin rod domain in cultured myotubes resulted in colocalization with, and eventual aggregation of, the desmin-containing IFs, but did not noticeably affect myofibril assembly and alignment. Rather than playing a direct role in myofibrillogenesis, it is likely that IFs play a role at the next level of cytoskeletal organization by preserving the overall shape and integrity of developing muscle cells as they grow into elongated myotubes.