The spinal cord contains neuronal networks that are involved in various important functions; including sensory, motor and autonomic controls. In 1988 Kosaka et al. reported on the co-localisation of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunoreactivity within the cervical regions of the rat spinal cord. The present study examines the distribution of ChAT immunoreactivity and co-localisation with green fluorescent protein (GFP) in the cervical, thoracic and lumbar cord sections of transgenic adult mice expressing GFP under control of the GAD67 promoter (GAD67-GFP mice) (Tamamaki et al., 2003). GAD67-GFP mice (4-6 weeks, n = 3) were injected intraperitoneally with 0.1 ml of 1 % Fluorogold. After 24-48 hours the mice were anaesthetised intraperitoneally with 60 mg/kg sodium pentobarbitone and when the pinch reflex was absent, they were perfused transcardially with 4 % paraformaldehyde. The spinal cords were postfixed with 4 % paraformaldehyde for 24 hours. The tissues were sectioned at 50 µm on a vibrating microtome and the sections were processed utilising double labelling immunofluorescence (IHC) for ChAT and GFP. Co-localisation of staining was determined for neurones per section and percentage co-localisation was determined from 10 sections from each animal for each spinal region.Neurones immunoreactive (IR) for ChAT or GAD67-GFP were found throughout the spinal cord. Co-localisation of GFP-IR and ChAT-IR was observed predominantly in lamina X (co-localised neurones per section being 4.03 ± 0.29 (mean ± SEM) for cervical, 3.80 ± 0.42 for thoracic and 4.47 ± 0.59 for lumbar sections). In lamina X GFP was co-localised with ChAT in 9.04% (121/1338) of GAD and ChAT neurones in the cervical sections, 8.14% (114/1400) in the thoracic sections and 9.76% (134/1373) in the lumbar sections. A limited number of co-localised neurones were also observed in the dorsal horn (co-localised neurones per section 1.13 ± 0.35 (mean ± SEM) for cervical, 0.43 ± 0.03 for thoracic and 0.67 ± 0.07 for lumbar sections) and lamina VII (co-localised neurones per section 0.20 ± 0.12 (mean ± SEM) for cervical and 0.10 ± 0.06 for lumbar sections). The co-localised neurones were negative for Fluorogold indicating that they are not motor or preganglionic neurones. In addition, based on a reconstruction of the thoracic cord serial sections, the majority of co-localised neurones were located ventral and ventrolateral to the central canal. Since the projections and functions of these co-localised neurones in lamina X have not been reported, it is worth studying further how they are involved in neuronal circuits.