Cytochrome b5/cytochrome b5 reductase (cb5/cb5r) is a cytosolic fusion protein between the hemoprotein cytochrome b5 and the flavoprotein cytochrome b5 reductase. We describe the identification and characterization of a novel splice variant of cb5/cb5r in the mouse and rat and show that expression of the variant is conserved in both species but is not expressed in human tissue. Characterization of the exon structure of cb5/cb5r indicated that the variant was due to the deletion of the whole of exon 12, thus the variant was named cb5/cb5rΔ12. Exon 12 codes for the flavin-adenine dinucleotide binding domain of cb5/cb5r. Expression analysis revealed the transcript of cb5/cb5rΔ12 in mouse and rat testis, brain, and skeletal muscle and also in the male germ line. We postulate that cb5/cb5rΔ12 may function in a dominant negative fashion, limiting the amount of damage caused by the production of reactive oxygen species by cb5/cb5r.