Voltage gated potassium channel subunits of the Kv3 subfamily (Kv3.1-Kv3.4) play a vital role in action potential repolarisation in neurons (Rudy and McBain). We have previously reported on the differential distribution of Kv3.1b, Kv3.2 and Kv3.4 (Brooke et al., 2004) in the medulla oblongata and spinal cord. Since cellular and sub-cellular differences in the pattern of ion channel expression can aid in defining specific roles of channels, here we examine the distribution of the Kv3.3 subunit. Rats (100-200g, n=5) were injected intraperitoneally with 0.1ml 1% Fluorogold (Fluorochrome Inc.) and 7 days later were humanely killed by Sagatal (100mg/kg i.p) and perfused transcardially with 4% paraformaldehyde/0.1-0.5% glutaraldehyde. Sections (50 m) of medulla and thoracic spinal cord were cut and incubated in anti-Kv3.3 antibody (Alomone; 1:1K), followed by a Cy3 conjugated secondary antibody and dual labelled with various antibodies to neurochemical markers, visualised using a biotinylated conjugated secondary antibody and Streptavidin Alexa488. Kv3.3-immunoreactivity (Kv3.3-IR) was observed in the somata of discrete neuronal populations throughout the spinal cord and medulla. Particularly dense labelling was observed in motor nuclei, dorsal column nuclei (DCN), dorsal medullary nucleus, lateral reticular nucleus, spinal trigeminal nucleus (SpV). A few strongly labelled cells were present in the medial subnucleus of the nucleus tractus solitarius (NTS), raphe nuclei and throughout spinal laminae III-X. Kv3.3-IR neurones did not contain 5-HT or tyrosine hydroxylase, but some cells co-localised with the 200Kd neurofilament, NF200, a marker for heavily myelinated neurones. Kv3.3 predominantly co-localised with Kv3.1b, but some Kv3.1b-IR cells did not contain Kv3.3-IR. Autonomic preganglionic nuclei were predominantly devoid of labelling. Kv3.3-IR was identified in presynaptic terminals throughout the brainstem and spinal cord by co-localisation with SV2 and/or electron microscopic examination. These terminals were immunopositive for markers of excitatory (VGluT2) or inhibitory (GlyT2) terminals. This study indicates that Kv3.3 subunits are found in neuronal somata and presynaptic terminals throughout the spinal cord and brainstem. The Kv3 subunits are differentially distributed, suggesting specific functions in some cell types.