The X-ray structure of benzoylformate decarboxylase (BFDC), from P. putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from L. lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from S. cerevisiae which indicated that it might be active as a dimer. To investigate this possibility in BFDC we sought to shift the equilibrium towards dimer formation. Point mutations were made in the non-catalytic monomer-monomer interfaces but these had minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E/A306F, were sufficient to markedly alter the dimer-tetramer equilibrium, and to provide an ~450-fold decrease in kcat value. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site as well as to the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.