The subplate plays an important role in forming neuronal connections during early cortical development. We characterized by the use of whole-cell and cell-attached patch-clamp recordings in coronal brain slices from newborn mice (postnatal day [P] 0-3) the functional properties of two major pathways onto subplate neurons (SPn), the thalamocortical and the intra-subplate synaptic input. The two afferent pathways were stimulated extracellularly with bipolar electrodes placed in the thalamus and the subplate, respectively. Synaptically evoked and pharmacologically isolated N-methyl-d-aspartate receptor (NMDAR) -mediated responses with an onset latency of approximately 6 ms could be reliably recorded in P0-3 SPn. Whereas the intra-subplate input revealed a pronounced facilitation using paired pulse stimulation at 60-120 ms or repetitive activation at 10-40 Hz, the thalamocortical input was either stable or markedly suppressed under these conditions. Single cell reverse transcription PCR revealed the expression of the NR2A, B and D subunit in all investigated SPn. The intra-subplate and the thalamocortical synaptic input did not differ in their sensitivity to NVP-AAM077 or ifenprodil, indicating that both synaptic inputs have a similar NR2A/2B subunit composition. At P0, NMDAR-mediated synaptic inputs arising from the thalamus were significantly larger as compared with the intra-subplate input. This difference could no longer be detected in P2-3 SPn, indicating an input-specific developmental regulation during the first Ps. Our data indicate that the thalamocortical and intra-subplate synaptic input onto P0-3 SPn differs in functional, molecular and developmental properties. The intra-subplate synaptic input shows more mature functional properties and sustains high stimulation frequencies, thereby promoting the immature thalamocortical input to the developing neocortical circuit.