Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillogenesis. Several developmental stages of these cultures were double stained with antibodies to titin in combination with antibodies to alpha-actin, alpha-actinin, myosin heavy chain (MHC), nebulin, desmin, and beta-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing alpha-actinin and sarcomeric alpha-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, alpha-actin and alpha-actinin. Thereafter a more periodic localization of titin, MHC, alpha-actin and alpha-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and alpha-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.