The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO(2), 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed to mediate this reaction catalysed by an iron-sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron-sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases and the related benzoyl-CoA reductases-present in anaerobically living bacteria-employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.