The supramolecular assembly of Helicobacter pylori urease was studied by nanoflow electrospray ionization orthogonal time-of-flight mass spectrometry. The measured molecular mass of the urease complex of 1.06 MDa corresponds to a dodecameric (αβ)12 assembly of urease α (26 kDa) and β (61 kDa) subunits. The dodecamer disassembles readily into (αβ)3 subunits in solution and under controlled collisional-induced dissociation in the gas phase. This is in strong support of an ((αβ)3)4 architecture consistent with the recently published x-ray structure. In vitro, the α and β subunits are capable of re-assembling to (αβ)3, but not further to the dodecameric complex. Copyright © 2003 John Wiley & Sons, Ltd.