When human erythrocyte apo-transketolase is mixed with cofactors and substrates, the progress curve exhibits a lag phase. Elimination of the lag phase requires the presence of saturating concentrations of cofactors, thiamin diphosphate and Mg2+. The most simple explanation of the observed hysteretic transition is that the slow binding of a Mg2+-thiamin-diphosphate species precedes slow isomerisation of the enzyme to the active form. Although the hysteretic transition involves more than one process, it does not involve the dissociation–association of enzyme subunits. The best estimate of the apparent Km, 1.59 ± 0.23 μM, for the binding of Mg2+-thiamin diphosphate to transketolase was obtained in the presence of a high non-inhibitory concentration of magnesium and varied concentrations of thiamin diphosphate. Thus the reconstitution of the human enzyme differs from the yeast enzyme, which undergoes a rate-limiting dimerisation during reconstitution.