Aldose reductase, which catalyzes the reduction of glucose to sorbitol as part of the polyol pathway, has been implicated in the development of diabetic complications and is a prime target for drug development. However, aldose reductase exhibits broad specificity for both hydrophilic and hydrophobic aldehydes, which suggests that aldose reductase may also be a detoxification enzyme. Several series of structurally related aldehydes were compared as substrates in order to deduce the structural features that result in low Michaelis constants. Aldehydes that contain an aromatic ring are generally excellent substrates, consistent with crystallographic data which suggest that aldose reductase possesses a large hydrophobic substrate binding site. However, there is little discrimination among different aromatic aldehydes. In addition, small hydrophilic aldehydes exhibit low Km values if the alpha-carbon is oxidized. Analysis of the binding of NADPH by fluorescence quenching techniques indicates that aldose reductase exhibits higher affinity for NADPH than NADP, suggesting that this enzyme is normally primed for reductive metabolism. Thus aldose reductase appears to have evolved to catalyze the reduction of a very broad range of aldehydes. Structural features of substrates that bind to aldose reductase with low Km values were used to identify potential endogenous substrates. 4-Hydroxynonenal, a reactive alpha-beta unsaturated aldehyde produced during oxidative stress, is an excellent substrate (Km = 22 microM, kcat/Km = 4.6 x 10(6) M-1 min-1). Reductive metabolism of endogenous aldehydes in addition to glucose, catalyzed by aldose reductase, may play an important role in the development of diabetic complications.