The reaction mechanism of the F1 isozyme of horse liver aldehyde dehydrogenase (EC 126.96.36.199) was investigated using both steady-state and rapid kinetic techniques. Using the steady-state substrate velocity patterns, the NADH inhibition patterns at several aldehyde concentrations, and the substrate analog (adenosine diphosphoribose and chloral hydrate) inhibition patterns, the enzymic catalysis was shown to involve ordered addition of NAD followed by aldehyde. This mechanism was confirmed using the kinetics of the hydrolysis of p-nitrophenyl acetate as an indicator of the dehydrogenase substrate binding. Steady-state experiments with deuteroacetaldehyde showed the V to be unchanged, but the Km increased ( Km CH3CDO Km CH3CHO = 3.4). Stopped flow experiments where E-NAD was rapidly mixed with aldehyde showed a burst of NADH formation followed by slower steady-state turnover. This result clearly indicates that the rate limiting step lies after NAD reduction. The NADH off rate (0.7 s-1) as estimated by displacement of NADH from the E-NADH complex upon rapid addition of NAD was found to be very close to the steady-state site turnover number (0.3 s-1). This fact and the relatively small effect of aldehyde R-group on maximal velocity suggest that the slow rate of NADH release contributes significantly to limitation of the enzyme catalytic velocity.