The purpose of this study was to examine the role of the two high molecular weight proteins, titin and nebulin, in skeletal muscle myofibrillogenesis. A monoclonal antibody against chicken breast titin was produced, and used in conjunction with polyclonal antibodies against chicken breast titin and nebulin, and monoclonal antibodies to actin and muscle-specific myosin in a series of double-label immunofluorescence studies to examine the organization of these proteins in cultured skeletal muscle cells derived from 12 day chick embryos. Postmitotic, mononucleated myoblasts synthesized titin, myosin, actin, and nebulin. Initial myofibril assembly events involved the coalescence of titin and myosin in a continuous pattern along actin containing structures morphologically similar to stress fibers. Titin and myosin organization into the skeletal muscle myofibril appeared to be coupled, as adult-like titin banding patterns were not observed in the absence of myosin organization into A bands. Titin and actin organization, in contrast, did not appear to be linked. Organization of titin into adult-like double bands along nascent myofibrils consistently preceded that of actin. Neither nebulin and myosin nor nebulin and titin assembly appeared to be linked, since the appearance of nebulin banding was seen after the formation of titin and myosin bands along nascent myofibrils. Nebulin organization occurred either at the same time or after the assembly of actin into I bands. A possible correlation between the formation of adult-like nebulin double bands and the appearance of phase-dense Z lines was noted, however. Labeling experiments were repeated with cultures exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS). The results of labeling experiments of EMS cells confirmed those found in the normal culture system. In conclusion, it appears that titin does not play a scaffolding or template role for subsequent thick filament alignment into A bands. The possibility of a template role for thin filament alignment into I bands can not be excluded, however, because titin organization precedes actin. The relationship between nebulin organization and the appearance of Z lines suggests that nebulin may be involved in Z line assembly or may regulate the insertion of thin filaments into Z lines.