Synaptic activity in early postnatal life is important for the acquisition of mature structural and functional properties of neurons. Previous studies indicate that the mature molecular features of spinal motor neurons emerge during a period of activity-dependent development in early postnatal life. Since glutamatergic synaptic transmission provides the major excitatory drive into motor neurons, glutamate receptors are likely to play a central role in motor neuron activity-dependent development. To gain insight into this process, we have used receptor autoradiography, immunoblotting and immunohistochemistry to determine the distribution, temporal expression and potential subunit composition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors in the developing rat spinal cord. Using two different ligands, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and [3H]-6-cyano-7-nitroquinoxaline-2,3-dione, we find that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites in the adult are largely restricted to the substantia gelatinosa. In marked contrast, during early postnatal life, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites are transiently expressed at high levels in the ventral horn. This parallels previous findings on the developmental regulation of N-methyl-D-aspartate receptor expression. Using alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit-specific antibodies we show by immunoblot analysis and immunohistology that, to varying degrees, the expression patterns of glutamate receptor subunit 1 and glutamate receptor subunits 2/3 are significantly developmentally regulated. The most conspicuous change is the downregulation of glutamate receptor 1 expression within motor neurons over the first three weeks of postnatal life. The qualitative and quantitative changes we observe in glutamate receptor expression in early postnatal life are likely to have a major impact on the electrophysiological properties of young motor neurons and thus may contribute to their activity-dependent development.