The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possesshydrogenosomes.Hydrogenosomes arehighly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the ®rst time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a signi®cant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.