Dorsal horn interneurons in midlumbar segments are one of the major subpopulations of interneurons relaying information from group II muscle afferent fibres in spinal reflex pathways (Jankowska et al 2002). We used a combination of electrophysiological and immunocytochemical methods to study the axonal projections of these interneurons and the neurotransmitter content of their terminals.Adult cats (5) were deeply anaesthetised with chloralose (up to 50 mg kg-1 i.v.), following induction with sodium pentobarbital (40 mg kg-1 i.p.). Neuromuscular transmission was blocked (Pavulon 0.2 mg kg-1). All experimental procedures complied with national guidelines, including methods for ensuring anaesthesia was maintained during neuromuscular block, and can be found in Bannatyne et al (2003). Interneurons to be analysed were located in laminae IV-V, where stimulation of muscle nerves at intensities consistent with activation of group II muscle afferent fibres evoked maximal dorsal horn field potentials. Interneurons that were monosynaptically activated from these afferents were labelled intracellularly with rhodamine dextran and Neurobiotin. At the end of experiments the spinal cord was fixed by perfusion and the animals killed humanely. Sections containing axonal processes of 7 interneurons (to date) were incubated with antibodies raised against the following molecules: vesicular glutamate transporters 1 and 2 (VGLUT1 and 2) which are markers for glutamatergic terminals; glycine transporter molecule 2 (GlyT2) or gephyrin to reveal glycine-containing axons; or glutamic acid decarboxylase (GAD) to reveal GABAergic axons. Finally, labelled axons were processed for HRP histochemistry and reconstructed.Interneurons had axonal arbors in laminae IV-IX ipsilaterally and VI-VIII contralaterally. Immunocytochemical analysis of individual boutons showed that five neurons were glycinergic and two were glutamatergic. The population thus includes both excitatory and inhibitory cells. Our results confirm that this subpopulation of interneurons form boutons ipsilaterally in laminae VII and VIII where they may provide monosynaptic input to commissural cells interposed in pathways from group II afferents (Edgley et al 2003). They may also contact other groups of interneurons both ipsi- and contralaterally and form monosynaptic contacts with ipsilateral motorneurons; this is currently under investigation.