Juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol phosphotransferase (JHDPT) were characterized from the Malpighian tubules of day 1 fifth instar Manduca sexta. An improved RP-HPLC assay is described for the major metabolites of (lOR,lIS) juvenile hormone I: diol, acid, aciddiol, and diol-phosphate. JHEH is strictly associated with membrane fractions, while JHDPT is cytosolic. JHEH may be solubilized in active form by the nonionic detergents Thesit or MEGA-8. Separation of Malpighian tubule cytosol proteins using preparative isoelectric focusing yields two zones which contain JHDPT activity, at pi 4.8-5.1 and 6.8-8.2. The partially purified JHDPT from either zone requires both ATP and Mg2+ for activity, so this enzyme may be formally called either ATP:juveni/e hormone diol phosphotransferase or juvenile hormone diol kinase (EC 2.1.7.3). Metabolites more polar than JH I aciddiol and JH I diol-phosphate are generated in vivo from either [3H]JH I or 13H]JH I diol. 0 1995 Wiley-Liss, inc.