A biotinylated acetyl-CoA carboxylase from the microaerophilic bacterium Helicobacter pylori was partially purified and characterized. The approximate molecular mass of the native enzyme was estimated at 235 kDa by native PAGE. A single band corresponding to approximately 24 kDa was detected by SDS-PAGE, suggesting that the native enzyme is a multi-protein complex. The protein was isolated from the soluble fraction of the cell. Catalytic activity was acetyl-CoA-dependent and inhibited by avidin but unaffected by avidin pre-treated with excess biotin. The end-product of the reaction was identified as malonyl-CoA and the reaction was shown to be reversible by NMR spectroscopy. The activity of the enzyme was 0.29 μmol min^-1 (mg protein)^-1. The V _max for bicarbonate was calculated at 0.73 μmol min^-1 (mg protein)^-1, and the affinity of the enzyme for this substrate was relatively low, with an apparent K _m of 16.6 mM. These data provide the first evidence of a possible physiological role for the requirement of high levels of CO₂ for growth in vitro of this bacterium.