Thiazole synthase (ThiG) catalyzes an Amadori-type rearrangement of 1-deoxy-D-xylulose-5-phosphate (DXP) via an imine intermediate. In support of this, we have demonstrated enzyme-catalyzed exchange of the C2 carbonyl of DXP. Borohydride reduction of the enzyme DXP imine followed by top-down mass spectrometric analysis localized the imine to lysine 96. On the basis of these observations, a new mechanism for the biosynthesis of the thiazole phosphate moiety of thiamin pyrophosphate in Bacillus subtilis is proposed. This mechanism involves the generation of a ketone at C3 of DXP by an Amadoritype rearrangement of the imine followed by nucleophillic addition of the sulfur carrier protein (ThiS-thiocarboxylate) to this carbonyl group.