The decarboxylation of glutaconyl-CoA to crotonyl-CoA in the anaerobic bacterium Acidaminococcus fermentans is catalysed by a membrane-bound, biotin-dependent enzyme which requires Na+ for activity. Inverted vesicles from A. fermentans accumulated Na+ only if glutaconyl-CoA was decarboxylated. The Na+ uptake was inhibited by avidin but not by the avidin biotin complex. Detergents and ionophores such as monensin also prevented the Na+ transport. The results indicate that the enzyme is able to convert the free energy of decarboxylation (ΔG o′ ≈−30 kJ/mol) into a Na+ gradient.