The mechanism of reduction of p-nitrosophenol (pNSP) catalyzed by horse liver alcohol dehydrogenase (HADH) and human pi-alcohol dehydrogenase (pi-ADH) has been compared in transient and steady-state experiments. Our results indicate that pNSP reduction catalyzed by these two ADH proceeds by different mechanisms. In one mechanism, shown by Equation 1, pNSP is reduced to p-aminophenol (pAP) via two enzymatic steps (Steps 1 and 3), which are mediated by the nonenzymatic dehydration of p-N-hydroxyaminophenol (pN-OHAP) to 1,4-benzoquinoneimine (BQI) (Step 2). [formula: see text] Pathway (I) is proposed mainly for pi-ADH but can be catalyzed by HADH. However, Step 3 is catalyzed approximately 2 orders of magnitude more slowly by HADH than by pi-ADH. This conclusion is confirmed by the results, which indicate that pi-ADH very efficiently catalyzes the reduction of BQI and 1,4-benzoquinone (BQ) to the corresponding hydroquinones. The kinetic constants determined at pH 7.4 suggest that pi-ADH is a more efficient quinone reductase and nitroso reductase than it is an ethanol oxidase or acetaldehyde reductase. An alternative mechanism of pNSP reduction, shown by Equation 2, is suggested for HADH. In this mechanism, formation of the p-hydroxybenzylnitrenium ion (pNH+P) occurs at the active-site zinc ion of the enzyme (Step 2) and accelerates further nonenzymatic reduction to pAP or hydrolysis to BQ (Step 3). [formula: see text]