The kinetic mechanism of a long-chain aldehyde dehydrogenase that is induced during the development of bioluminescence in Beneckea harveyi has been investigated. Parallel lines were obtained in Lineweaver-Burk plots with NAD+ and long-chain aldehydes (heptaldehyde, nonylaldehyde). However, product and dead-end inhibitor studies, substrate protection (NAD+, aldehyde) against inactivation with N-ethylmaleimide, and in particular, a noncompetitive substrate inhibition pattern with aldehyde at high concentrations showed that aldehyde dehydrogenase had a sequential mechanism. The data were consistent with a nonrapid equilibrium random mechanism with a preferred order of addition of substrates (NAD+, aldehyde) and an ordered release of products with NADH release being the last and rate-limiting step in the reaction, a mechanism very similar to that found for short-chain mammalian aldehyde dehydrogenases. The present experiments emphasize the caution that must be taken in interpreting parallel patterns in initial velocity experiments, as well as the difficulty in classifying sequential enzyme mechanisms as either strictly ordered or random.