Development of primary cultured chicken myogenic cells were studied using living cell micro-morphoanalysis, muscle specific protein immunofluorescent double staining, image projection analysis and 3H-TdR incorporation autoradiography methods. Changes in single, normal newborn myoblasts from the time of their last mitosis until 22 hr old were followed. All +/- 4 hr myoblasts were desmin+ and most were positive for alpha-actinin, zeugmatin, troponin-I (TnI), alpha-actin. titin, nebulin and myosin heavy chain (MHC). There was no obligatory temporal or spatial sequence in the order of the appearance of the major myofibrillar proteins. Nascent sister myoblasts assumed an exceptionally elongated bipolar morphology that is as singular to mononucleated postmitotic myoblasts as is their capacity to transcribe myofibrillar genes. The assembly of non-striated myofibrils (NSMFs) was evident in all 6-8 hr cells and was initiated in the absence of myomesin and C-protein. Myomesin first appeared along NSMFs in 10-14 hr old cells. C-protein was only found localized to transverse doublets bisecting 1.6 microns wide A-bands of assembled sarcomeres. Each newly assembled sarcomere presented the same invariant distribution of proteins that is found in adult sarcomeres. There is a lag of 16 or more hours between the first appearance of most of the major myofibrillar proteins and their assembly into NSMFs and the first appearance of striated myofibrils (SMFs). The observations indicated that the majority of normal myoblasts up-regulate the synthesis of myofibrillar proteins prior to, not after, fusion. In brief, new-born +/- 4 hr myoblasts expressed their differentiation program in the process as (1) withdrawal from the cell cycle: (2) initiation of synthesis and accumulation of desmin and 7 early myofibrillar proteins: (3) cellular elongation and assembly of NSMFs and SMFs: (4) acquisition of a fusion-competent sarcolemma. The expression of this cell autonomous myogenic differentiation program is distorted or blocked in rhabdomyosarcoma RD cells. The majority of RD cells expressed desmin (50-90%): among these desmin+ cells, 10-20% incorporated 3H-TdR. In addition, 60-78% of the mitotic cells were desmin+. Most desmin+ cells were myofibrillar protein negative. Only a small number of tumor cells (5-10%) expressed MHC, titin, alpha-actinin and s-alpha-actin. 3H-TdR positive nuclei were observed in these myofibrillar protein+ cells: 11-12% in titin+ or nebulin+ cells and 4% in MHC+ cells. But none of the mitotic cells were myofibrillar protein+.(ABSTRACT TRUNCATED AT 400 WORDS)