A highly fluorogenic synthetic substrate for human "low K," aldehyde dehydrogenase (ALDH) isozymes, 7-methoxy-I-naphthaldehyde, was identified. The new substrate is characterized by submicromolar or low micromolar Michaelis constants (K,) for both cytosolic and mitochondrial ALDH from the human liver. The cytosolic ALDH oxidizes the naphthaldehyde substrate with a rate almost equal to that of the respective acetaldehyde reaction, while the mitochondrial isozyme is ca. 25 fold less active. Thanks to a high fluorescence yield of the oxidation product, i.e., the corresponding naphthoate (4 = 0.41), reaction rates as low as 0.1 nM min-' can be measured reproducibly. The emission spectral characteristics of the naphthoate product differ significantly from that of the aldehyde (A,, at 395 and 475 nm, respectively), allowing a selective observation of the former, and there is also marked difference between the naphthoate and the corresponding alcohol (A,, at 35.5 nm), helping to eliminate possible interferences from alcohol dehydrogenase and/or aldehyde reductase in the fluorimetric assay. As a possible application of the new assay, it is shown that the ALDH activity can easily be detected, by continuous monitoring of the fluorescence increase, in lOOO-fold diluted hemolysates, using 7-methoxy-1-naphthaldehyde and NAD+ as substrates, and there is no necessity for prior removal of hemoglobin. Normal ALDH level in the blood of healthy volunteers has been measured, and the result, 4.9 U 1-l (n = 27), agrees with the literature data obtained for the acetaldehyde oxidation activity, and exceeds by more than 20-fold the lower detection limit of the method. The present method is free of a background drift, characteristic of all the previously proposed spectral assays for ALDH.