1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD + -dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation ( a ), NAD + –Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD + was attached by using dicyclohexylcarbodi-imide; for preparation ( b ), AMP–Sepharose, N 6 -(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD + yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5 m . A 0.0–0.15 m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5m m -NAD + ); 80 (lactate+NAD + ); 95 (lactate+semicarbazide+NAD + ); 100 (0.5m m -NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.