SummaryThis investigation examined factors that affect methylenetetrahydrofolate (methylene-H4PteGlu) reductase activity in cultured human cells. Activity was demonstrable in extracts of cultured normal human skin fibroblasts, amniotic fluid cells, and lymphoblasts. The velocity of the reaction was maximal at pH 6.3 in extracts of all three cell types. Subcellular localization studies indicated that 83–94% of the reductase activity was extranuclear in the three cell types. The reductase activity was not substantially altered by growth of lymphoblasts whether in Eagle's minimum essential medium (MEM) supplemented with homocysteine alone or with additional hydroxocobalamin and folic acid, or in media containing a 5-fold greater concentration of methionine. In normal fibroblasts the methylene-H4PteGlu reductase activity varied little when the confluent cells were exposed to media deficient in or supplemented with varying amounts and combinations of methionine, homocysteine, folic acid, and cobalamin. Under our conditions the addition of purifiedS-adenosylmethionine to give a final concentration of 48 mM in the assay reaction mixture reduced the reductase activity to 38% of control in the fibroblast extract and to 13% of control in the extract of rat liver. This sensitivity to inhibition byS-adenosylmethionine and the lack of response to varied levels of methionine, homocysteine, folic acid, and cobalamin are further evidence that the reductase activities in mammalian liver and fibroblasts are similar.SpeculationCultured human skin fibroblasts and peripheral blood lymphoblasts may provide a useful, convenient system for studying the biochemical-genetic regulation of 5,10-methylene-H4PteGlu reductase activity and, ultimately, of the levels of its folate product, 5-methyl-H4PteGlu.