Neurotrophic factors are thought to be critically involved in formation and maintenance of the neuromuscular system. To know precise expression levels of these factors in the muscles during the postnatal period, we developed competitive RT-PCR and two-site enzyme immunoassay and quantitatively measured neurotrophic factors in the rat gastrocnemius and soleus muscles during the postnatal development. mRNAs of glial cell line-derived neurotrophic factor (GDNF) in the gastrocnemius and the soleus muscles were expressed in the highest amount among the neurotrophic factors at birth and dramatically decreased in the first 3 months, while GDNF proteins substantially existed at 3 months of age. Neurotrophin-3 and brain-derived neurotrophic factor in the gastrocnemius muscle kept constant expression in mRNA and protein during the postnatal period. In contrast, mRNA of neurotrophin-4 increased in the first 2 weeks. In the soleus muscles all the neurotrophic factor proteins increased with age for the first month, contrasting with their expressions in the gastrocnemius. The present results showed that GDNF is constitutively supplied to the neuromuscular junction (NMJ) during postnatal development and into adulthood, suggesting its importance in maintenance of the NMJ. Expression of other neurotrophins was also regulated independently during development possibly according to their own roles in the neuromuscular circuit.