SummaryMethylenetetrahydrofolate (methylene-H4PteGlu) reductase activities in extracts of both normal and reductase-deficient cells were low and quite variable during the logarithmic phase of growth. Higher activities were detected reproducibly when the cultures were confluent. Methylene-H4PteGlu reductase activities in extracts of fibroblasts from the parents of patientCPwere about half of the level observed in normal control subjects. In fibroblasts from patientCP, the activity was 20% of normal whereas, from patientsLMandBM, the activities were 19% and 14% of normal, respectively. When incubated at 55° in a solution containing all the components of the standard reaction mixture except the 5-methyl-H4PteGlu substrate, the reductase activity in extracts of fibroblasts from two unrelated normal control subjects decreased to 31% and 22%, respectively, of the initial values after 30 min of incubation. In contrast, the reductase in extracts of cells patientCPwas rapidly and exponentially inactivated at 55°. The reductase activity in extracts from patientsLMandBM, the sisters, decreased to 22% and 38%, respectively, of the initial values. In repeated experiments the heat inactivation of reductase activity in extracts of cells fromLMandBMclosely resembled the normal control subjects in total extent and time course of inactivation. The reductase activity in extracts fromWMa, a fourth, unreleated patient was also completely inactivated but somewhat less rapidly than with patientCP.These results provide strong evidence that the reductase deficiency in these three families is due to different alleles. The data suggest that inCPandWMathere is a mutationally induced structural defect in the aporeductase as the basis for the observed alteration in thermostability, presumably reflecting reduced ability to bind the FAD cofactor.SpeculationThe observation that methylene-H4PteGlu reductase activity in crude fibroblast extracts from two of the three families studied shows reduced thermal stability as well as decreased activity raises the possibility that this enzyme is unusually susceptible to mutationally induced alterations in cofactor binding.