Sulfurospirillum multivorans is an epsilonproteobacterium able to grow anaerobically with hydrogen as electron donor and tetrachloroethene (PCE) as terminal electron acceptor (organohalide respiration). The dechlorination is mediated by the PCE reductive dehalogenase (PceA), which is a corrinoid-containing iron-sulfur protein [1]. This enzyme is located at the periplasmic face of the cytoplasmic membrane [2] and represents the terminal oxidoreductase of a so far uncharacterized membrane-associated respiratory chain. The corrinoid cofactor of PceA was shown to be Norpseudo-B12. However, until now, only little is known about the role of the iron-sulfur clusters in the electron transfer within the enzyme. In this study a mutant strain of S. multivorans producing an affinity-tagged PceA was characterized with regard to growth and the ability to dechlorinate PCE as terminal electron acceptor. A simplified protocol for PceA purification was established and optimized for a fast and efficient isolation of pure and homogenous recombinant PceA in high amounts. The iron content and the number of corrinoids per molecule PceA were quantified. Using electron paramagnetic resonance spectroscopy (EPR) and concomitant redox titration, the type of the Fe-S clusters and their midpoint redox potentials were determined. In addition, the midpoint redox potentials of the corrinoid cofactor were measured and compared to already published data [3]. From the experiments presented here a tentative scheme of the electron transport pathway in the PCE respiratory chain of S. multivorans was derived that combines recent biochemical and spectroscopical results.