A cDNA encoding a GABAA receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human ε-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588–3595] that the rodent ε-subunit mRNA encoded an additional sequence (≈400 residues). We provide evidence that human and rat ε-subunit are similar in size. The distribution of cells expressing the GABAAε-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that ε-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat ε-subunit revealed the presence of ε-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, α3, θ and ε GABAA subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABAA receptors may regulate, neuroendocrine and modulatory systems in the brain.