To determine the relationship between thin filaments, Z-bands, microtubules, intermediate filaments (IFs), T-tubules, and sarcoplasmic reticulum (SR) during myofibrillogenesis, myotubes were selectively depleted of their myofibrils with 12-tetradecanoylphorbol 13-acetate (TPA) and then were allowed to regenerate in (i) normal medium, (ii) taxol, and (iii) Colcemid. Myofibrils assembled in normal medium formed typical A-, I-, Z-, M-, and H-bands and associated IFs, T-tubules, and SR. Myofibrils assembled in taxol formed "A-bands" of aligned thick filaments interdigitating with long microtubules and "I-bands" consisting only of microtubules. These unprecedented sarcomeres lacked thin filaments, Z-bands, and associated IFs and SR. "Solitary A-bands," consisting exclusively of laterally aligned bipolar thick filaments 1.6 microM in length without either thin filaments or microtubules, were observed. Myofibrils assembled in Colcemid formed all myofibrillar components in the absence of microtubules but these did not achieve rigorous lateral alignment. Colcemid and taxol induced the formation of patchy Z-bands that invariably served as insertion sites for thin filaments, irrespective of the presence or absence of adjacent thick filaments. Z-bands may function as actin-organizing centers for each sarcomere.