The two major purposes of these studies were to examine: (1) the intrinsic cellular differences expressed by myogenic cells derived from fast- and slow-growing animals and (2) the appearance, accumulation and organization of nebulin, a recently discovered myofibrillar protein, in skeletal myogenesis;In the first area of study, muscle cell cultures derived from genetically fast- (fast cultures) and slow- (slow cultures) growing chickens and grown under identical conditions exhibited significant (p < 0.01) differences in proliferation, protein accumulation, insulin response and insulin binding. The fast cultures exhibited a larger number of fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to addition of insulin to the culture medium as reflected by increased fusion rate and protein accumulation at 24 h in culture. Slow cultures bound more ('125)I-insulin than fast cultures at 24 h in culture. These results demonstrated that the growth potentials of fast- and slow-growing animals are due to intrinsic cellular differences in early myogenic cells;Nebulin, a component of the N(,2)-line in skeletal and cardiac muscle myofibrils (Wang and Williamson, Proc. Natl. Acad. Sci. 77:3254, 1980), was isolated from chicken skeletal muscle myofibrils and used for preparation of antibodies. Indirect immunofluorescence localization demonstrated that nebulin was present in an N-line-like arrangement in adult skeletal myofibrils and in cryostat sections of adult skeletal muscle tissue. In both cardiac and skeletal muscle cultures, immunofluorescence localization of nebulin progressed from a light diffuse fluorescence pattern in early myogenic cells to a fluorescence pattern similar to that seen in adult myofibrils in skeletal myotubes and cardiac cells that had assembled myofibrils. Immunoprecipitation analysis of ('35)S-methionine labeled skeletal muscle cultures demonstrated a three-fold increase in nebulin accumulation during the period of greatest fusion activity. The ratio of nebulin plus titin, another high molecular weight protein, to other myofibrillar proteins was higher in embryonic myofibrils than in adult myofibrils. These results demonstrated that nebulin is accumulated at the same time during differentiation as other myofibrillar proteins and is incorporated into myofibrils during their assembly and not later.