Two forms of NADH-cytochrome b5 reductase (b5R), soluble and membrane-bound, are known. A hypothesis that the human soluble form b5R is generated through post-translational processing of the membrane-bound form was previously proposed. In this study, the 5'-rapid amplification of cDNA ends for human reticulocyte, liver, brain, and HL-60 cell mRNAs revealed the ubiquitous presence of an alternative type of human b5R mRNA which can probably be translated into the soluble form b5R directly; however, the erythroid-specific transcript of the b5R gene was not found. This type of b5R mRNA initiating from at least two sites contains a non-coding new first exon located between the first two exons of the human b5R gene identified before. In addition, this new first exon shares 62% homology with the first exon and its 3'-flanking intron sequences of rat erythroid-specific b5R mRNA, whereas the 5'-flanking region of the new first exon possesses features of house-keeping gene. These results might be important to understand the regulation mechanism of human b5R biosynthesis and divergent evolution of the gene regulation.