Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A human cDNA sequence [J. Xuan, J. Kowalski, A.F. Chambers, and D.T. Denhardt (1994) Genomics 20, 129-131] containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases [F.M. Hahn and C.D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The human cDNA sequence was cloned into expression plasmid pFMH12. The encoded protein was overproduced in Escherichia coli and purified to > 90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography. The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+. The Michaelis constant for IPP was 33 microM, similar to the value of 43 microM reported for yeast IPP isomerase; Vmax = 4.1 mumol min-1 mg-1 for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme [I.P. Street and C.D. Poulter (1990) Biochemistry 29, 7531-7538].