In the neocortex, the effectiveness of potential cellular repopulation therapies for diseases involving neuronal loss may depend critically on whether newly incorporated cells can differentiate appropriately into precisely the right kind of neuron, re-establish precise long-distance connections, and reconstruct complex functional circuitry. Here, we test the hypothesis that increased efficiency of connectivity could be achieved if precursors could be more fully differentiated toward desired phenotypes. We compared embryonic neuroblasts and immature murine neurons subregionally dissected from either embryonic day 17 (E17) (Shin et al., 2000) or E19 primary somatosensory (S1) cortex and postnatal day 3 (P3) purified callosal projection neurons (CPNs) with regard to neurotransmitter and receptor phenotype and afferent synapse formation after transplantation into adult mouse S1 cortex undergoing targeted apoptotic degeneration of layer II/III and V CPNs. Two weeks after transplantation, neurons from all developmental stages were found dispersed within layers II/III and V, many with morphological features typical of large pyramidal neurons. Retrograde labeling with FluoroGold revealed that 42 +/- 2% of transplanted E19 immature S1 neurons formed connections with the contralateral S1 cortex by 12 weeks after transplantation, compared with 23 +/- 7% of E17 neurons. A greater percentage of E19-derived neurons received synapses (77 +/- 1%) compared with E17-derived neurons (67 +/- 2%). Similar percentages of both E17 and E19 donor-derived neurons expressed neurotransmitters and receptors [glutamate, aspartate, GABA, GABA receptor (GABA-R), NMDA-R, AMPA-R, and kainate-R] appropriate for endogenous adult CPNs progressively over a period of 2-12 weeks after transplantation. Although P3 fluorescence-activated cell sorting-purified neurons also expressed these mature phenotypic markers after transplantation, their survival in vivo was poor. We conclude that later-stage and region-specific immature neurons develop a mature CPN phenotype and make appropriate connections with recipient circuitry with increased efficiency. However, at postnatal stages of development, limitations in survival outweigh this increased efficiency. These results suggest that efforts to direct the differentiation of earlier precursors precisely along specific desired neuronal lineages could potentially make possible the highly efficient reconstruction of complex neocortical and other CNS circuitry.