Two forms of NADH-cytochrome b(5) reductase (b(5)R), soluble and membrane-bound, are known. A hypothesis that the human soluble form b(5)R 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 b(5)R mRNA which can probably be translated into the soluble form b(5)R directly; however, the erythroid-specific transcript of the b(5)R gene was not found, This type of b(5)R mRNA initiating from at least two sites contains a non-coding new first exon located between the first two exons of the human b(5)R 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 b(5)R 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 b(5)R biosynthesis and divergent evolution of the gene regulation. (C) 1997 Academic Press.