In contrast to aneurally cultured human muscle, which is immature in regard to its morphologic phenotype and only rarely and weakly contracts spontaneously, innervated cultured human muscle fibres have: (1) nearly continuous, d-tubocurarine-inhibitable contractions; (2) well-developed cross-striations, basal lamina, t-tubules, and postsynaptic folds of the neuromuscular junctions; (3) the majority of their nuclei peripheralized; and (4) acetylcholinesterase-positive sites present only at the neuromuscular junctions. To see whether the expression of the muscle morphologic phenotype is induced only by neural factors generated from the spinal cord explants or also by their frequent contractile activity, we paralyzed innervated cultured human muscle fibres with 2 microM tetrodotoxin for four weeks, either from the first day of muscle contractions or following four weeks of muscle contractions. In both experimental designs, by light microscopy tetrodotoxin paralysis abolished cross-striations and caused prominent internalization of muscle nuclei; however, it did not influence the intensity of acetylcholinesterase staining at the neuromuscular junctions. By electron microscopy, there was no difference between paralyzed and contracting muscle fibres in development of t-tubules, basal lamina and postsynaptic folds. Our study demonstrates that in human muscle contractile activity: (1) regulates peripheral migration of nuclei and development of cross-striations; and (2) does not influence development of the neuromuscular junction, basal lamina, and t-tubules, which are mainly regulated by neural influences. This culture model may be useful for studying detailed mechanisms of human muscle fibre development and structural abnormalities in human neuromuscular diseases.