The development and adult production of birdsong are subserved by specialized brain nuclei, including the robust nucleus of the archistriatum (RA), and its afferents originating in the caudal nucleus of the ventral hyperstriatum (HVc) and the lateral portion of the magnocellular nucleus of the anterior neostriatum (L-MAN). An in vitro brain slice preparation was used to characterize the electrophysiological properties of L-MAN and HVc axonal synapses within RA and to examine how these synaptic connections change during the course of song development. Electrical stimulation of L-MAN and not HVc fibers evoked excitatory synaptic potentials from virtually all RA neurons in brain slices prepared from male and female zebra finches less than 25 d of age. These “L-MAN” EPSPs were blocked substantially by the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-APV; 50–100 microM) and by hyperpolarization of the postsynaptic membrane. In contrast, when slices were prepared from male finches greater than 35 d of age, electrical stimulation of the L-MAN and the HVc fiber tracts evoked synaptic responses from over 70% of RA neurons. Although the L-MAN EPSPs resembled those seen in RA before day 25, the “HVc” EPSPs were relatively insensitive to D-APV, but almost completely abolished by 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA glutamate receptor antagonist. These experiments indicate that L-MAN and HVc axons make pharmacologically distinct synapses on the same RA neurons, and that these synapses first form at different stages during development.