The inhibitory neurotransmitter GABA plays an important role in regulating the activity of magnocellular oxytocin and vasopressin neurons located in the supraoptic and paraventricular nuclei through occupancy of GABAA receptors. However, the GABAA receptor is a hetero-oligomeric protein comprised of different subunits and the subunit types expressed in a given receptor complex appear critical for its sensitivity to GABA, benzodiazepines and/or steroids. Thus, in order to understand fully the GABAergic control of oxytocin and vasopressin secretion, definition of the GABAA receptors synthesized by magnocellular neurons in the supraoptic and paraventricular nuclei is required. In the supraoptic nucleus, antibodies directed against the alpha 1, alpha 2 and beta 2/3 subunits of the GABAA receptor revealed similar strong antigen distribution on all magnocellular neurons. Using sequential double-immunoperoxidase staining, immunoreactivity for all three subunits was observed on both oxytocin and vasopressin neurons of the supraoptic nucleus. In contrast, only alpha 2 subunit immunoreactivity was detected on the cell bodies of oxytocin and vasopressin neurons in the paraventricular nucleus. No sex differences were detected. In situ hybridization experiments using 35S-labelled oligonucleotides showed that all supraoptic neurons expressed alpha 1, alpha 2 and beta 2 subunit messenger RNA transcripts while magnocellular neurons in the paraventricular nucleus were only enriched in alpha 2 subunit messenger RNA. Quantitative analysis showed that the expression of alpha 1 and beta 2 subunit messenger RNAs in the paraventricular nucleus was half that observed in the supraoptic nucleus while expression of beta 3 subunit messenger RNA was very low in both nuclei. These results show that all oxytocin and vasopressin neurons located in the supraoptic nucleus synthesize and express alpha 1, alpha 2 and beta 2 subunits of the GABAA receptor while those in the paraventricular nucleus are only immunoreactive for the alpha 2 subunit. These observations suggest, therefore, that at least two pharmacologically distinct GABAA receptor isoforms exist on supraoptic neurons and that these are different to those expressed by paraventricular magnocellular cells. Thus, in addition to providing a definition of the subunits likely to form specific GABAA receptor isoforms on magnocellular neurons, this study gives direct evidence for GABAA receptor heterogeneity between supraoptic and paraventricular neurons, but not between oxytocin and vasopressin cells.