Earlier studies from this laboratory showed that human folylpolyglutamate synthetase (FPGS) exists as cytosolic and mitochondrial (mFPGS) isoforms. Localization of mFPGS within mitochondria may help elucidate how the enzyme functions to maintain the mitochondrial folate pool. A human T-lymphoblastic leukemia CCRF-CEM cell lysate was fractionated by differential centrifugation into cytosolic and mitochondrial fractions. Activity assays for cytosol-and mitochondria-specific enzymes verified the purity and integrity of the fractions. Mitochondria were subfractionated with increasing concentrations of digitonin to successively extract the four submitochondrial compartments. Western analyses of the fractions using protein markers specific for each compartment suggest that mFPGS is distributed in the matrix and/or inner membrane compartments. Further support for an interaction of mFPGS with the inner mitochondrial membrane is provided by localization of about half of the mFPGS in the mitochondrial membrane fraction obtained by freeze-thaw of intact mitochondria; the remaining mFPGS is located in the soluble fraction. Resistance of about half of the mFPGS in whole mitochondria to alkaline carbonate extraction suggests that its interaction with the inner membrane is more similar to an integral, than a peripheral, membrane protein. The data suggest that human mFPGS is at least in part strongly associated with the inner mitochondrial membrane.