The neuronal organization of the spinal cord, and the elucidation of properties that permit the integration of proprioceptive, descending and intrinsic segmental neural activity to produce movement, have challenged neuroscientists for decades. The excitation and inhibition of motoneurones during locomotor activity is controlled by spinal interneurones. Classical in vivo experiments have led to the classification of interneurones by their location, segmental input, and axonal projections (Jankowska, 1992). However, using these criteria it has not been possible to provide a comprehensive classification scheme that unambiguously defines the functional roles and properties of ventral horn interneurones. The generation of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in specific populations of interneurones has provided the methodology that enables the identification and study of spinal interneurones. In Hb9-GFP mice, eGFP is expressed under the control of the murine promotor of the homeodomain protein HB9. These mice (Wichterle et al., 2002) carry an estimated 510 copies of a transgene comprised of a ~9 kb fragment with the 5’ upstream region of the murine HB9 gene followed by a 5’ splice substrate, an eGFP gene, and a polyadenylation signal. In these Hb9-GFP mice, some ventral interneurons, in addition to motoneurons express GFP. Of note are a discrete, clustered population of GFP+ interneurons which are located in medial lamina VIII throughout the spinal cord, and which, unlike other GFP+ interneurons, are indeed Hb9 positive. Electrophysiological and anatomical investigations were carried out on this genetically defined population of interneurones.To assess whether the medial Lamina VIII cells are involved in locomotor activity, adult mice were subjected to a 90 minute overground locomotor task. Following an intraperitoneal injection of ketamine and intracardiac perfusion, the spinal cords were removed, post-fixed and immunohistochemically processed for the activity-dependent immediate early gene c-fos. Double labeling of c-fos and GFP in the medial Lamina VIII cells indicates that these interneurones are active during locomotion. To assess the transmitter phenotype of these cells, we used fluorescent in situ hybridization with a mRNA probe for the vesicular glutamate transporter vGlut2. Data revealed that the eGFP+ medial lamina VIII interneurones are glutamatergic. To identify the electrophysiological properties of these cells, we obtained whole cell patch clamp recordings from medial lamina VIII eGFP+ neurones. Experiments were done at a developmental stage where the mice could weight bear and walk with their abdomens pendant (> postnatal day 8). To obtain spinal cord slices, mice were anaesthetized with ketamine, decapitated and their spinal cords removed. 200 µm thick slices of the upper lumbar spinal cord were cut on a vibrating microtome and equilibriated for an hour prior to recording. GFP positive interneurons in medial lamina VIII were identified and patch-clamped using IR-DIC optics and epifluorescence. Two distinct electrophysiological classes of neurones were evident. The first class (n= 40) consisted of neurones with very small somata (mean ± sdev: 9.3 ±1.4 pF), high input resistance (1012 +/- 321 MΩ) and a prominent post-inhibitory rebound (PIR) giving rise to action potential doublets (100%). The PIR, a property important in rhythm-generating neurones, is mediated by a nickel-sensitive transient calcium current. Many of these cells (58%) were spontaneously firing. The second class of Lamina VIII GFP positive interneurons (n=8) were larger (16.8 ± 2.2 pF, p < 0.002, student’s t-test), and had lower input resistances (520 ± 200 MΩ, p < 0.002), prominent hyperpolarisation-activated inward currents (Ih, 100%). These cells had spontaneous biphasic oscillations of membrane potential characteristic of electrotonic coupling (100%).This study demonstrates that advances in developmental biology combined with genetic technology provide the methodology to identify classes of spinal interneurones and to study their anatomical and physiological properties. This is necessary for the characterisation of spinal motor networks.