Voltage-gated sodium channels participate in the propagation of action potentials in excitable cells. Eukaiyotic Nays are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1-S6). The first four segments form the voltage-sensing domain and S-5 and S-6 create the pore domain with the selectivity filter. Prokaryotic Nags resemble these characteristics, but are truly tetrameric. They can typically be efficiently synthesized in bacteria, but production in vitro with cell-free synthesis has not been demonstrated. Here we report the cell-free expression and purification of a prokaryotic tetrameric pore-only sodium channel. We produced milligram quantities of the functional channel protein as characterized by size-exclusion chromatography, infrared spectroscopy and electro-physiological recordings. Cell-free expression enables advanced site-directed labelling, post-translational modifications, and special solubilization schemes. This enables next-generation biophysical experiments to study the principle of sodium ion selectivity and transport in sodium channels. (C) 2015 The Authors. Published by Elsevier Inc.