Human class III alcohol dehydrogenase (with both glutathione-dependent formaldehyde dehydrogenase and alcohol dehydrogenase activities) was expressed, and studied by site-directed mutagenesis corresponding to three amino acid residues that are affecting the substrate-binding pocket of class I (with alcohol dehydrogenase activity only). A Thr48Ala exchange results in an enzyme essentially without alcohol dehydrogenase activity but with some glutathione-dependent formaldehyde dehydrogenase activity retained. This indicates that coordination to the enzyme of S-hydroxymethylglutathione is mediated by interactions additional to, or different from, those utilized for primary and secondary alcohols. An Asp57Leu mutation causes considerable loss of the formaldehyde dehydrogenase activity, showing that a negative charge at position 57 is a prerequisite for this class III-type of activity, in the same manner as a positive charge at position 115 has been previously demonstrated to be crucial. Therefore, Asp57 and Arg115 appear to contribute equally to the interactions with S-hydroxymethylglutathione, compatible with defining the class III-type of specificity and possibly explaining the dependence on glutathione. A Tyr93Phe mutant exhibits decreased kcat values for substrates in general and correlates with inhibition of alcohol dehydrogenase activity by 4-methylpyrazole, a potent inhibitor of the class I enzymes. In a double mutant, Asp57Leu/Tyr93Phe, the effects of the two mutations are potentiating one another, yielding a fall in kcat/Km for hydroxymethylglutathione by a factor of 1250, i.e., a still further loss of class III-type activity.(ABSTRACT TRUNCATED AT 250 WORDS)