1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR, also known as methyl-D-erythritol 4-phosphate (MEP) synthase) is a NADPH-dependent enzyme, which catalyzes the conversion of DXP to MEP in the nonmevatonate pathway of isoprene biosynthesis. Two mechanisms have been proposed for the DXR-catalyzed reaction. In the cl-ketol rearrangement mechanism, the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-migration to give methylerythrose phosphate, which is then reduced to MEP by NADPH. In the retroaldol/aldot rearrangement mechanism, DXR first cleaves the C3-C4 bond of DXP in a retroaldol manner to generate a three-carbon and a two-carbon phosphate bimolecular intermediate. These two species are then reunited by an aldol reaction to form a new C-C bond, yielding an aldehyde intermediate. Subsequent reduction by NADPH affords MEP. To differentiate these mechanisms, we have prepared [3-H-2]- and [4-H-2]-DXP and carried out a competitive secondary kinetic isotope effect (KIE) study of the DXR reaction. The normal 2 degrees KIES observed for [3-H-2]and (4-H-2]-DXP provide compelling evidence supporting a retroaldol/aldol mechanism for the rearrangement catalyzed by DXR, with the rate-limiting step being cleavage of the C3-C4 bond of DXP.