Intracellular Cl- plays a key role in cellular volume regulation, cell cycle control and shaping the polarity of inhibitory postsynaptic responses mediated by anion-permeable GABA and glycine receptors. In this study, we have investigated the expression patterns of members of the cation-chloride cotransporters (CCCs), including the K-Cl cotransporters KCC1-4 and the Na-K-2 Cl cotranporter NKCC1 during rodent embryonic brain development. At the time of neurogenesis (embryonic days; E12.5-14.5), KCC1 was only detectable in the developing choroid plexus. KCC2 mRNA was detectable as early as E12.5 in the ventral part of the (cervical) spinal cord, and by E14.5, the expression had spread to TUJ1-positive differentiating regions of the rhombencephalon, diencephalon and olfactory bulb, in parallel with neuronal maturation. KCC3 mRNA was scarce in the cortical plate at E14.5, and slightly up-regulated at birth. In contrast, KCC4 mRNA was abundantly expressed in the ventricular zone and was down-regulated perinatally. At E14.5, NKCC1 was highly expressed in the vimentin-positive radial glia of the proliferative zone of the subcortical region. At later embryonic stages, during gliogenesis (E17-P0), there was a shift in NKCC1 expression to the neuron specific Class III beta-tubulin (betaIII) positive region of the cortical plate. These unique spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl- regulatory mechanisms are critically involved in the control of neuronal development.