Cystathionine beta-synthase has been purified from human liver more than 3000-fold by a series of steps including high speed centrifugation, ammonium sulfate fractionation, chromatography on hydroxylapatite and DEAE-cellulose, gel filtration, preparative polyacrylamide gel electrophoresis, and glycerol density gradient centrifugation. The enzyme obtained is homogeneous as judged by polyacrylamide gel electrophoresis in four different systems: native, isoelectric focusing, in sodium dodecyl sulfate, and in 8 M urea. The native enzyme has an estimated molecular weight of 94,000 and is composed of two apparently identical subunits of 48,000. The pure enzyme has a specific activity of 160 units/mg of protein and contains tightly bound cofactor, pyridoxal 5' -phosphate. It is possesses serine sulfhydrase as well as cystathionine synthase activity. It has a broad pH optimum from 8.4 to 9.0, apparent Km values for L-serine of 1.15 mM and for L-homocysteine of 0.59 mM, and a pI of 5.2 The enzyme is stable over a pH range from 6.5 to 8.0 in phosphate buffers and can be stored in 40% glycerol at -15 degrees C for at least 1 month.