Laminar specificity is one of the most striking features of neocortical circuitry. To explore the molecular basis of this specificity, particularly in relation to thalamocortical connectivity, we searched for the genes expressed in the upper cortical layers by constructing a subtraction cDNA library that was enriched for genes expressed in layer 4 of perinatal rat somatosensory cortex. Differential screening, sequence analysis and in situ hybridization demonstrated that a new unc5 family member (unc5h4), deltex-like gene, stem cell factor (SCF) and myocyte-specific enhancer factor-2C (MEF-2C) were specifically expressed in layer 4 or layers 2/3-4 at postnatal day 7, by when laminar organization and fundamental cortical circuitries have been established. In terms of regional specificity, unc5h4 and SCF signals were stronger in sensory cortices, whereas MEF-2C and deltex-like gene were expressed rather uniformly in all neocortical regions. Analysis during development demonstrated that expression of these genes was pronounced between late embryonic and early postnatal developmental stages, except for MEF-2C expression, which continued in later stages. These results demonstrate that certain types of molecules including transcription factors, receptor and ligand molecules, are expressed specifically in the upper layers of the developing neocortex, suggesting a role in laminar specification of cortical cells and circuitry.