4-trans-(N,N-Dimethylamino)cinnamaldehyde (DACA) is a chromophoric substrate of aldehyde dehydrogenase (EC 1.2.1.3) whose fate can be followed during catalysis. During this investigation we found that DACA also fluoresces and that this fluorescence is enhanced and blue-shifted upon binding to aldehyde dehydrogenase. Binding of DACA to aldehyde dehydrogenase also occurs in the absence of coenzyme. Benzaldehyde (a substrate), acetophenone (a substrate-competitive inhibitor), and the substrate-competitive affinity reagent bromoacetophenone interfere with DACA binding. Thus, DACA binds to the active site and can be employed for titration of active aldehyde dehydrogenase. Both E1 and E2 isozymes, which are homotetramers, bind DACA with dissociation constants of 1-4 microM with a stoichiometry of 2 mol DACA/ mol enzyme. The stoichiometry of enzyme-acyl intermediate was also found to be 2 mol DACA/ mol enzyme for both E1 and E2 isozymes. Thus, both enzymes appear to have only two substrate-binding sites which participate in catalysis. The level of enzyme-acyl intermediate remained constant at different pH values, showing that enhancement of velocity with pH was not due to altered DACA-enzyme levels. When the reaction velocity was increased even further by using 150 microM Mg2+ the intermediate level was decreased, suggesting that both increased pH and Mg2+ promote decomposition of the DACA-enzyme intermediate. Titration with DACA permits study of aldehyde substrate catalysis before central complex interconversion.