Abstract Coenzyme A-linked aldehyde dehydrogenase from Escherichia coli strain B was purified 170-fold over cell-free extracts, and certain of its properties were investigated. The enzyme is essentially inactive in the absence of sulfhydryl compounds such as dithiothreitol and β-mercaptoethanol. Addition of a thiol reagent after incubation of the enzyme with substrates results in very slow reactivation. Incubation of aldehyde dehydrogenase with DPN plus mercaptan before addition of other substrates is required in order to obtain normal initial velocities. The kinetics of the system was investigated from both sides of the reaction. Initial velocity experiments suggest the mechanism is "ping-pong." These studies, although still preliminary, imply that acetyl-CoA adds to the dehydrogenase before addition of DPNH. With regard to the other side of the reaction, it is thought that DPN and acetaldehyde interact with the enzyme prior to the addition of CoA. The last step in the reaction sequence may be a transacetylation between the enzyme and CoA to form acetyl-CoA. Additional properties of the enzyme, such as optimum pH, substrate specificity, and reaction stoichiometry, were also studied.