The gene encoding trypsin-solubilized bovine liver microsomal cytochrome b5 (82 residues in length) has been mutated, in which the codons of Glu44 and Glu56 were changed to those of Ala. The mutated genes were expressed in Escherichia coli successfully and three mutant proteins (E44A, E56A and E44/56A) were obtained. The UV-visible, CD and 1H NMR spectra of proteins have been studied. The results show that the mutagenesis at surface residues does not alter the secondary and tertiary structures of cytochrome b5 significantly. The interactions between recombinant cytochrome b5 and its mutants with cytochrome c were studied by using optical difference spectra. The results demonstrated that both Glu44 and Glu56 of cytochrome b5 participate in the formation of a complex between cytochrome b5 and cytochrome c.