An expression vector for NADH-cytochrome b5 reductase containing a thrombin cleavage site directly before the N-terminal glycine residue of the flavoprotein was used to isolate the non-myristoylated enzyme by thrombin cleavage of the initial fusion protein of a short segment of the multiple cloning site of the plasmid vector and the reductase. This flavoprotein preparation, containing only the 28-residue N-terminal peptide segment of the membrane-binding domain of the mammalian enzyme, binds to phospholipid vesicles and interacts with membrane-bound cytochrome b5. The effect of N-myristoylation of the enzyme therefore appears to be limited to facilitating and stabilizing interactions with phospholipid vesicles. However, the relatively short intervening peptide sequence that separates the crucial peptide membrane-binding domain from lysine 41, which has been implicated in the active-site interaction with cytochrome b5 (Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) J. Biol. Chem. 267, 2519-2523), provides some limitation of the distance from the membrane surface for the interactions required for rapid electron transfer from the flavin of the reductase to the heme of cytochrome b5.