The expression of the proenkephalin (PEnk) gene in rat neocortex develops during the first two postnatal weeks in an outside first-inside last mode that is opposite to the gradient of neurogenesis. To test whether the distribution of PEnk gene expression depends on the formation of the local circuitry, we examined the role of glutamate neurons in the expression of the gene in slice cultures of rat frontoparietal cortex. In situ and Northern blot hybridization were used for analysis. In slices explanted at postnatal day 6, the neuronal expression of the PEnk gene developed as in vivo. The expression responded to glutamate receptor agonists and antagonists in a time-dependent manner. After 2 days in vitro the expression of the gene was only enhanced by N-methyl-D-aspartate receptors, whereas after 7 days in vitro AMPA receptors also regulated the expression. We concluded that glutamate neurons are involved in the development and maintenance of the PEnk gene expression in the neocortex.