GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl^-]_i in immature neocortical neurons causes GABA_A receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular ' accumulation remain controversial. Therefore, the GABA reversal potential (E_GABA) or [Cl^-] _i in early postnatal rat neocortical neurons was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation- Cl^- cotransporter mRNAs (in the same cells) were examined by semi-quantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl^-]_i. The mRNA expression levels were positively (the Cl^--accumulating Na⁺, K⁺-2Cl^- cotransporter NKCC1) or negatively (the Cl^--extruding K⁺-Cl^- cotransporterKCC2) correlated with [Cl^-]_i. NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl^-]_i and NKCC1 mRNA expression were each higher in cortical plate (CP) neurons than in the presumably older layer V/VI pyramidal neurons in a given slice. The pharmacological effects of bumetanide on E_GABA were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex.