To illustrate the functions of the aromatic residue Phe35 of cytochrome b₅ and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b₅, we studied electron transfer reactions of cytochrome b₅ and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)]^-, [Fe(CDTA)]^- and [Ru(NH₃)₆]³⁺. The changes at Phe35 of cytochrome b₅ and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.