Histidine at position 51 of the class I beta 1 alcohol dehydrogenase (ADH) functions as a general base by indirectly abstracting a proton from the alcohol substrate through a hydrogen-bonded proton relay system. The human class II pi-ADH was reported to be polymorphic, having either Ser or Thr, but not His at position 51. It is unknown whether Ser or Thr51 have a catalytic role in ethanol oxidation with pi-ADH. Accordingly, we expressed and purified recombinant mutants of pi-ADH with Thr, Ser, and His at position 51. At pH 6.5, values for Vmax/Km for ethanol were 0.30, 0.10, and 0.09 min-1 mM-1 for pi 51Thr, pi 51Ser, and pi 51His ADH, respectively. Hence the effects of the substitutions were much less than the 11-fold decrease in Vmax/Km observed for beta 1-ADH when a neutral amino acid (Gln) was substituted for His51. Addition of a buffer base (400 mM glycylglycine) had little effect on Vmax/Km of recombinant pi 51Thr or pi 51Ser ADH, while it increased Vmax/Km for ethanol 7-fold for the beta (1)51 Gln ADH. We conclude that there is no evidence for Thr51 of pi-ADH participating in a proton relay similar to that seen in beta 1-ADH and that a base at position 51 may not be a universal requirement for a functional alcohol dehydrogenase with a moderate efficiency for ethanol oxidation at a physiological pH.