Titin is amongst the first sarcomeric proteins to be detected in the process of myofibrillogenesis of striated muscle. During embryogenesis this high molecular weight protein is initially observed in a punctate staining pattern in immunohistochemical studies, while during maturation titin organizes into a cross-striated pattern. The dynamic process of titin assembly up to its integration into the sarcomeres of cultured human skeletal muscle cells has been studied in subsequent stages of differentiation with antibodies to four well-defined titin epitopes. Since in maturated muscle cells these epitopes are clearly distinguishable on the extended titin molecule we wondered how these epitopes reorganize during myofibrillogenesis, and whether such a reorganization would reveal important clues about its supramolecular organization during development. Immunofluorescence staining of postmitotic mononuclear myoblasts indicate that the investigated epitopes of the titin molecule are displayed in a punctate pattern with neighboring, but clearly separate spots in the cytoplasm of the cells. During elongation and fusion of the cells, these titin spots associate with stress fiber-like structures to finally reach their position at either the Z-line, the A-I junction or the A-band. We propose that during this transition the large titin molecule is unfolded, with the amino terminus of the molecule migrating in the direction of the Z-line and the carboxy terminus moving towards the M-line. In maturated, fused myotubes the final cross-striated patterns of all investigated titin epitopes are observed. While this process of unfolding of the titin molecule progresses, other compounds of the Z-line and the A-band migrate to their specific positions in the nascent sarcomere. A-band components such as sarcomeric myosin and C-protein, are also observed as dot-like aggregates during initial stages of muscle cell differentiation and organize into a cross-striated pattern in the sarcomere virtually simultaneously with titin. The Z-line associated component desmin organizes into a cross-striated pattern at a later stage.