Complex I (NADH:ubiquinone oxidoreductase, NUO) and complex I-like enzymes have been detected in several organohalide-respiring bacteria at genomic and proteomic levels. The question of the involvement of these enzymes in organohalide respiration remains to be elucidated. Comparative physiology and proteomics were applied to study the role of the 11-subunit complex I-like enzyme in Desulfitobacterium hafniense strain DCB-2, a model organism able to conserve energy via organohalide respiration (OHR). It appears that the complex I-like enzyme is generally used in the energy metabolism of strain DCB-2 (and not specifically during OHR), that it becomes essential when organic electron donors such as lactate are used, where it may act as the entry point in the membrane cytoplasmic side for the electron transfer chain.