Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiaeΔfre1Δfre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe3+). The expression of each of these cytochromes b561 was able to rescue the growth defect of the Δfre1Δfre2 mutant cells in iron-deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3+-EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site-directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate-dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b561 family of proteins.