To investigate the unknown stereochemical course of the reaction catalyzed by the type-II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2-13 C 2 ]-IPP stereospecifically labelled with 2 H at C(2) was prepared by incubating a D 2 O solution of (E)-4-hydroxy-3methyl[1,2-13 C 2 ]but-2-enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13 C-NMR spectroscopy, treatment of the deuterated IPP with either type-I or type-II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2 H label of the starting material. From the known stereochemical course of the type-I isomerase-catalyzed reaction, one has to conclude that the label introduced from D 2 O in the course of the IspH reaction resides specifically in the H Si ÀC(2) position of IPP and that the two isomerases mobilize specifically the same H Re ÀC(2) ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D 2 O with the type-II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)-methyl group of DMAPP.