Two new fluorogenic substrates for human alcohol dehydrogenase (ADH), 4-methoxy-1-naphthaldehyde (IA) and 6-methoxy-2-napthaldehyde (IIA), are described. The 2-naphthaldehyde derivative fluoresces in aqueous media with a quantum yield of 0.22 with an emission maximum at 450 nm, but the 1-naphthaldehyde shows only weak fluorescence. The corresponding alcohol reduction products, 4-methoxy-1-naphthalenemethanol (IB) and 6-methoxy-2-naphthalenemethanol (IIB), exhibit fluorescence in the near uv region with quantum yields of 0.36 and 0.26, respectively. The Km values for the individual homogenous class I ADH isozymes, with the above naphthaldehydes as substrates, range from 0.35 to 11.5 microM. The kappa cat values range from 70 to 610 min-1 and are thus comparable to those for the best ADH substrates. Except for the beta 1 beta 1 isozyme, IA is the preferred substrate for class I ADH isozymes while IIA is rapidly reduced by class II (pi-ADH). The sensitivity and specificity of the enzymatic assay with IA as substrate are demonstrated and provide the basis for the determination of class I ADH activity in human serum.