The rat hepatic microsomal conversion of crotonyl- and hexenoyl CoA to butyrate and hexanoate was supported only by NADPH, while both NADH and NADPH were effective cofactors in the conversion of trans-2-hexadecenoyl CoA to palmitate. Experiments using mixtures of long- and short-chain enoyl-CoA substrates and competition experiments support the conclusion that microsomes contain 2 distinct enoyl CoA reductases, (1) a long chain enoyl CoA reductase capable of accepting reducing equivalents from either NADH or NADPH, and (2) a NADPH-specific short chain enoyl CoA reductase.