An NADPH-dependent aldehyde reductase was purified from rat brain microsomes to electrophoretic homogeneity. The purified enzyme had a molecular weight of 75, 000 and reduced long chain fatty aldehydes such as octanal and hexadecanal with higher affinity (K_m values of 0.21mM and 0.03mM, respectively) than for various artificial carbonyl compounds such as p-nitrobenzaldehyde and p-nitroacetophenone (K_m values of 0.31mM and 1.4mM, respectively). The purified microsomal aldehyde reductase also showed NADPH-cytochrome c reductase activity, and it could not be distinguished from NADPH-cytochrome c reductase in molecular weight (75, 000), chromatographic behavior, electrophoretic mobility, or immunological properties. The solubilized microsomal fraction treated with steapsin lost the reductase activity for hexadecanal but not that for cytochrome c. These results suggest that the aldehyde reductase in brain microsomes is identical to NADPHcytochrome c reductase and that a hydrophobic portion of the NADPH-cytochrome c reductase is required for the reduction of hexadecanal.