The nucleus of tractus solitarius (NTS) is the main integration site for autonomic control. It receives afferent information from arterial baroreceptors, chemoreceptors, cardiac receptors, gastrointestinal receptors and respiratory receptors (1). Details on the neurochemical properties of NTS neurones have been established by many researchers. However information on the co-localisation of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunoreactivity within the NTS has not been reported. This study examined the distribution of ChAT immunoreactivity and co-localisation with green fluorescent protein (GFP) in the NTS of transgenic mice (n=4) expressing GFP under control of the GAD67 promoter (GAD67-GFP mice) (2) using fluorescence immunohistochemistry (3). Four GAD67-GFP adult mice (mixed sexes) were injected intraperitoneally with 1 % Fluorogold. After 24-48 hours the mice were anaesthetised with 60 mg/kg sodium pentobarbitone and when the withdrawal reflex was absent, they were perfused transcardially with 4 % paraformaldehyde. The brainstems were postfixed with 4 % paraformaldehyde for 24 hours. Brainstems were sectioned (at 50 µm and 30 µm for double and triple labelling respectively). Serial sections were processed utilising fluorescent immunohistochemistry (IHC). One wild type adult mouse was also prepared in the same way as the GAD67-GFP mice. Sections were processed using diaminobenzidine IHC for ChAT to provide a permanent reaction product. ChAT immunoreactive (ChAT-IR) and GAD67-GFP immunoreactive (GAD67-GFP-IR) neurones were found in all NTS subnuclei. Moreover, co-localisation was observed in some NTS subnuclei; predominantly in the intermediate subnucleus (co-localised neurones per section were 6.83 ± 1.89 (mean ± SEM) for the right and 6.67 ± 2.19 for the left NTS column, n = 6 sections) and the central subnucleus (1.00 ± 0.68 (right NTS) and 2.17 ± 1.42 (left NTS)). Nitric oxide synthase immunoreactive (nNOS-IR) neurones in the central subnucleus are reported to be involved in oesophageal regulation (4). In our study, although nNOS-IR neurones were prominent in the central subnucleus, they did not exhibit co-localisation with either ChAT or GAD67-GFP. Although the absence of co-labelling in the central subnucleus with nNOS does not support a role for ChAT/GAD-IR neurones in oesophageal control, this possible function cannot be excluded at this time. Examination of other brainstem areas revealed co-localisation of ChAT and GAD67-GFP immunoreactivity, including the area postrema, the reticular formation and the lateral paragigantocellular nucleus. Since there is limited information of co-localisation for ChAT and GAD immunoreactivity in the brainstem, it is worth studying further to investigate its roles in these neurones.