The central nervous system plays is key to maintaining cardiovascular homeostasis e.g. plasma volume regulation, through the actions of the sympathetic nervous system. Recently it has emerged that activity present in the sympathetic nerves appears to be elevated in a number of cardiovascular diseases such as heart failure (Esler & Kaye). Functional studies indicate that within the PVN both GABA and nitric oxide (NO) play a role in determining the level of excitability of presympathetic neurones and that an alteration of this action may be in part responsible for the sympatho-excitation manifest in heart failure (Li & Patel, 2003). Anatomically, there is sparse evidence indicating how GABAergic and NO producing neurones might regulate presympathetic neurones. Therefore, the purpose of this study was to identify the location of GABAergic and NO producing neurones in the PVN in relation to a group of presympathetic neurones that play a role in plasma volume regulation. All procedures were carried out in accordance with the UK Animal (Scientific Procedures) Act 1986. Male Sprague Dawley rats were anaesthetised with Isofluorothane (4% in 2.5 l/min O₂) and the left adrenal gland isolated via a retroperitoneal approach. This was injected with 10 μl of herpes simplex virus conjugated with green fluoresecent protein (HSV-1-GFP; 10⁹ pfu/ml). The animals recovered for 4 days when they were killed humanely (Euthatal), perfused-fixed (4% paraformaldehyde) and the brain and spinal cord removed. Frozen sections (30 μm) containing virally labelled presympathetic neurones were incubated in either anti guinea pig GABA or anti guinea pig nNOS (neuronal NO synthase) for 24 hrs. The tissue was then treated with a biotinylated secondary antibody followed by strepavidin Alexa Fluor 350 or 594. The tissue was examined under epifluorescence. Presympathetic neurones containing HSV-1-GFP were scattered throughout the 5 parvocellular nuclei of the PVN, with the majority occupying the dorsal cap region. GABAeregic neurones were located at the lateral boundaries of the dorsal cap subnucleus and also intermingled with the presympathetic HSV-1-GFP neurones in this region. Nitric oxide (nNOS) containing neurones were found scattered through the magnocellular and parvocellular nuclei of the PVN and adjacent to the presympathetic neurones. We have provided evidence that within the PVN, GABA and NO containing neurones are found in close proximity to presympathetic neurones that project to the adrenal medulla. The close association of these pools of neurones provides an anatomical basis for the involvement of GABA and NO in modulating presympathetic neuronal excitability.