A large number of different unsaturated, saturated and hydroxylated aliphatic aldehydes can be generated during the peroxidation of cellular lipids. This study examined the kinetic properties of purified Class 3 rat aldehyde dehydrogenase (ALDH) with respect to the oxidation of various lipid aldehyde substrates. It also compared the substrate preference of the prototypic Class 3 ALDH with that of the constitutive rat microsomal aldehyde dehydrogenase. The results suggest that (1) microsomal ALDH is a member of the Class 3 aldehyde dehydrogenase family, and (2) the physiological role of the Class 3 ALDHs, including the microsomal form, is the oxidation of medium (6 to 9 carbon) chain length saturated and unsaturated aldehydes generated by the peroxidation of cellular lipids. Short chain aliphatic aldehydes, such as a malondialdehyde and 4-hydroxyalkenals, are not substrates for the Class 3 aldehyde dehydrogenases.