B-myc is a member of the myc gene family. Previous studies indicate that the rat B-myc gene contains a single exon which shows 77% nucleotide homology to the second exon of the rat c-myc gene. Its open reading frame (ORF) encodes a polypeptide with a predicted molecular weight of 20 kD. We have isolated a new, larger rat B-myc genomic clone. Sequence analysis of this clone confirmed the presence of one single coding exon. Furthermore, a genomic mouse B-myc clone was identified and compared to the rat homolog. Nucleotide analysis of B-myc coding and non-coding sequences suggests that it may be a functional gene evolved by selective duplication of part of the second c-myc exon. Analysis of the rodent B-myc open reading frames revealed two in-frame amino acid duplications in mouse B-myc and a 96% conservation at the amino acid level. Both rat and mouse B-myc proteins contain an identical and unique stretch of 14 carboxy terminal amino acid residues not found in other myc proteins. In vitro translation of rat and mouse B-myc ORF's yielded proteins that migrated as 26 kD bands in SDS-PAGE and could be immunoprecipitated by a polyclonal panmyc antiserum. Immunostaining of human lymphoma cells transiently transfected with a B-myc expression vector showed that the protein was mainly localized to the nucleus.