The membrane potential at endplates of the rat hemidiaphragm for 9-day-old rats increases by 1.8 mV after addition of D-tubocurarine. The endplate depolarization before the addition of D-tubocurarine is considered to be due to non-quantal release (NQR) of acetylcholine (ACh). In the presence of an anticholinesterase this depolarization increased. It was further enhanced by 0.1 -1.0 mM Mg2+ and reduced by 4 mM Mg2+ concentration. Thus the regulation of NQR at neuromuscular junctions of developing rat muscles is similar to that seen in adult mammalian species. The effect of NQR of ACh on neuromuscular contacts of muscle fibres from 8–9-day-old rat diaphragm and soleus muscles was studied. Pre-incubating the muscles in solutions where NQR was increased by lowering Mg2+ caused a significant (P < 0.01) reduction of neuromuscular contacts. This reduction did not occur when muscles were incubated in high Mg2+, when NQR is reduced. Increasing quantal release by high Ca2+ also caused a reduction of neuromuscular contacts. Histological examination of soleus muscle fibres treated with an anticholinesterase showed that muscles incubated in solutions with low (0.1 mM) concentrations of Mg2+ had significantly fewer neuromuscular contacts (38%) than those incubated in high concentrations of Mg2+ (61%). It is concluded that the NQR as assessed here contributes to the elimination of polyneuronal innervation during postnatal development of rat muscles.