The previously published procedure (Kraus et al. (1978) J. Biol. Chem.253, 6523–6528) for the purification of cystathionine β-synthase [l-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5′-phosphate-dependent enzyme from human liver has been modified. The new procedure, starting with a liver homogenate “aged” for 7 days at 4 °C, yielded homogeneous enzyme purified over 3000-fold with a much improved yield. “Aging” of the enzyme in crude homogenates yields a form apparently smaller by gel electrophoresis and with significantly increased activity and antigenicity. This species of cystathionine β-synthase does not form stable complexes with other proteins during purification as does the previously employed, freshly used species. An absorption spectrum and an amino acid composition of the pure enzyme were determined; the amino-terminal residue was shown to be methionine. The isoelectric points of holosynthase and aposynthase were estimated to be 5.2 and 5.6, respectively. Rabbit antiserum raised against the pure cystationine β-synthase was characterized using as antigen crude synthase from five different mammalian species as well as the pure human enzyme.