An affinity resin comprised of pyrazole bound to Sepharose 4B or polyacrylamide at the 4-position through a spacer arm of ≥12 Å is used to purify and fractionate crude liver alc. dehydrogenase (I) into a pyrazole-binding fraction and a pyrazole-insensitive fraction containing the anodic band of I. Thus, a crude solution of I is mixed with NAD, and the resulting complex is applied to the affinity resin. The bound fraction is eluted with EtOH; this fraction can be separated further into isoenzymes by adjusting the alc. gradient of the eluant. The pyrazole-insensitive fraction, which is not bound to the affinity resin, is purified further by (NH₄)₂SO₄ precipitation and chromatog. on AMP-agarose. From 100 g liver, 18 mg of pyrazole-insensitive I with a sp. activity of 1.1 IU/mg, overall yield of 34%, and 550-fold purification was obtained. On Na dodecyl sulfate-starch gel electrophoresis, the product showed a single band more anodic than the pyrazole-binding isoenzymes. The Zn content was 4.5 g-atom Zn/mol enzyme.