Methylenetetrahydrofolate reductase from pig liver has been purified to homogeneity, as judged by several criteria: (i) a single band with a subunit molecular weight of 77,300 following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; (ii) a molecular weight determined by amino acid analysis of 74,500 per flavin, in agreement with the subunit molecular weight; and (iii) constant specific activities in the peak fractions during the final chromatography step. The purified enzyme exhibits a typical flavoprotein absorption spectrum. Methylenetetrahydrofolate reductase is a minor constituent of pig liver, and to obtain homogeneous enzyme, a 32,000-fold purification must be accomplished. The preparation described herein attains such purification in 5 steps and with a 14% yield. The enzyme isolated in this fashion is active and stable, and contains a stoichiometric complement of FAD. The enzyme is reducible under anaerobic conditions by 5-deazaflavin/EDTA/light or by NADPH. Reduction of 1 mol of enzyme-bound FAD requires 1.1 mol of NADPH. The reduced enzyme can be reoxidized by (6-R)-methylenetetrahydrofolate, again with nearly 1:1 stoichiometry. Steady state kinetic measurements of the NADPH-methylenetetrahydrofolate oxidoreductase activity give parallel line double reciprocal plots. The turnover number per mol of enzyme-bound flavin is 1600/min under Vmax conditions. The spectrum of the enzyme-bound flavin is significantly perturbed by the binding of S-adenosylmethionine, a metabolite known to be an allosteric modulator of the enzyme.