The aim of this study was to establish a co-culture model of rodent neurons, striated muscle fibres and glial cells, in order to get a better understanding of nerve-muscle interactions. The first step was to determine the evolution of morphological and physiological properties of isolated adult rat flexor digitorum brevis muscle fibres maintained in vitro under various culture conditions: cultured alone or co-cultured, cultured on a substrate uncoated or coated with an extracellular matrix (Matrigel). Voltage-gated sodium channels (NaChs) and acetylcholine receptors (AChRs) were characterized using the patch-clamp technique or by cytochemistry. When the fibres were cultured for up to 3 weeks alone in suspension in DMEM supplemented with 10% fetal calf serum (FCS), clusters of AChRs and NaChs normally located in the postsynaptic membrane of striated fibres gradually disappeared from the synaptic region after 7 days under these culture conditions. Simultaneously, Na+ currents decreased at the endplate area and increased along the fibre as revealed by patch clamp. Fibres cultured on Matrigel in a medium supplemented with 10% FCS showed lower survival duration (10 days). After 4-5 days culture, fibroblastic cells proliferated. The major part of these cells (around 60%), positive for anti-desmin and/or anti-vimentin, were satellite cells. Other adherent cells were fibroblasts (only positive for anti-vimentin) and probably endothelial cells. The reduced survival of adult fibres in vitro was believed to be related to the proliferative activity of satellite cells. Patch clamp and/or cytochemistry showed that AChRs and NaChs remained clustered at the endplate area in the fibres cultured on Matrigel (at least up to 6 days). However, an increase in sodium currents was also observed along the fibre. In co-culture, the Na+ conductance, recorded from patches situated far from the endplate of fibres co-cultured with neurons isolated from the dorsal portion of the spinal cord of newborn rats, increased from 4.78 ± 0.86 nA V^-1 at D2 to 9.71 ± 2.91 nA V^-1 at D6 (P0.05) from the value found in suspended fibres (9.38 ± 1.97 nA V^-1) at the same culture duration. Now, the control value measured in co-culture at D2 was lower than the value observed in fibres of the same culture duration cultured in suspension (6.46 ± 1.07 nA V^-1) or on extracellular matrix (6.05 ± 2.46 nA V^-1); this was attributed to the effects of nervous released factors. Na+ currents increased at D6 when compared with D2 values in all conditions. These findings were consistent with the hypothesis of a neosynthesis of Na+ channels in isolated fibres, correlated with a decrease of channel density at the endplate especially in DMEM cultured fibres. The increase in the time constants of the Na+ current observed in co-culture is related to a greater increase in the population of TTX-resistant Na+ channels.