Both class I and class II alcohol dehydrogenase (ADH) activities are present in human serum. The contribution of each class can be measured using two class-specific, fluorogenic substrates, 4-methoxy-1-naphthaldehyde and 6-methoxy-2-naphthaldehyde. The former is highly selective for class I isozymes, especially those containing alpha or gamma subunits, whereas class II (pi) ADH preferentially reduces the latter. Selective inhibition of class I ADH by 4-methylpyrazole further increases the specificity. Specificity, accuracy, and precision of the assay for serum measurements have been determined. The activity of class I ADH in normal human serum is below the limit of detection of this method, i.e., less than 1.0 nM/min. The activity of class II ADH in normal individuals is 15 +/- 5 nM/min. In some patients values as high as 2100 nM/min are observed for class I, but in all instances, the amount of class II found was higher than that of class I ADH.