The transient expression of neuropeptide transmitters is a common feature of the developing cortex. We have now analysed the role of cortical afferents in shaping the neurochemical architecture of rat visual cortex using organotypic cultures. Deafferented cortex monocultures prepared from newborn rats reveal a constant NPY mRNA expression in 6–8% of all cortical neurons up to 90 days in vitro (DIV). In contrast, afferent thalamocortical and corticocortical axonal innervation elicits a progressive reduction in the percentage of NPY mRNA expressing neurons from initially 6–8% in 30DIV cocultures to 2–3% and 3–4% respectively in 60DIV cocultures, which is maintained for up to 90DIV. This phenotype restriction is not observed in only efferently connected corticocollicular cocultures. Further, axonal innervation does not change the percentage of GAD mRNA-expressing neurons, which remains at 13% in mono- and cocultures. When feeding thalamocortical cocultures with monoculture-conditioned medium between 3–20DIV followed by normal medium up to 60DIV, the phenotype restriction fails to occur in the cocultured cortex. We conclude that cortex-derived factors secreted into the medium by a monoculture suppress the phenotype-restricting capacity of the afferents, but only when present within the first 14DIV during the period of formation of axonal connections. To elucidate the nature of the cortex-derived factors, brain-derived neurotrophic factor was applied to the medium. When applied for the first 14DIV, it does not prevent the phenotype restriction from occurring. This suggests that epigenetic factors such as axonal innervation and cortex-derived factors other than brain-derived neurotrophic factor govern a phenotype decision in neocortical neurons during a period of molecular plasticity.