Immunochemical experiments and in vitro binding studies have revealed that titin/connectin, the elastic protein that spans the whole distance of a half-sarcomere, associates with several myosin-binding proteins of the sarcomeric A and M band. Two of these proteins, M-protein and myomesin, anchor titin in the region of the M band. A detailed molecular map describing the arrangement of titin, M-protein and myomesin in this part of the sarcomere was recently proposed. Furthermore, specific binding sites between the molecules were identified. How these polypeptides function in the assembly of the sarcomeric M band region has, however, remained unclear. Here we describe the distribution patterns of different epitopes recognized by newly developed antibodies against the extreme carboxyterminal portion of titin that is anchored in the M band, during the in vitro differentiation of human skeletal muscle cells. In contrast to a set of antibodies directed against Z band, I band and A band titin epitopes, anti-M band titin did not stain titin aggregates or titin in non-striated myofibrils (NSMF). The M band epitopes were only revealed in their characteristic sarcomeric locations, and were obviously not accessible in the non-striated part of nascent myofibrils, or during earlier developmental stages. We speculate that this phenomenon is associated with "immature" tertiary/quarternary structures of M band titin that avoid preliminary binding of M band proteins. In contrast to earlier observations on myofibrillogenesis in the mouse embryo, all the titin epitopes studied were simultaneously detected in their specific positions. Thus, sarcomere assembly in the widely used in vitro model systems seems to proceed at a much higher speed than in vivo. Similarly, myomesin and M-protein were only perceptible in striated myofibrils. While myomesin antibodies stained myofibrils at the time-point of appearance of the first titin striations, the incorporation of M-protein was found to be slightly delayed. In several myotubes no expression of M-protein was observed even during mature stages. These observations indicate its less important general role in the process of myofibrillogenesis. Furthermore, the relative number of M-protein negative myotubes varied in cultures derived from different muscles. This confirms the observation that cultured satellite cells are predestined to form a certain type of myofibers.