Southern blot analysis of mouse genomic DNA reveals two EcoRI fragments which faintly hybridize to mouse Adh-1 cDNA and are not part of the Adh-1 gene. These fragments were isolated from agarose gels, cloned, and characterized. Sequence analysis of the 2.1-kb EcoRI fragment suggests that it is likely a pseudogene since it does not contain a long open reading frame. However; the 2.0-kb EcoRI fragment contains a coding sequence with a long open reading frame which corresponds to exon 6 of the mouse Adh-1 gene. Comparison of the coding sequence with other known ADHs suggests that the sequence has diverged sufficiently from any currently known class of ADH to be a possible distinct class. Further confirmation awaits analysis of currently available genomic clones. Using these sequences as probe, restriction fragment length polymorphisms were identified for each sequence between C57Bl/6J and DBA/2J inbred mouse strains. The strain distribution pattern for these allelic differences was determined among the B x D recombinant inbred strains. This analysis revealed that the 2.1-kb EcoRI sequence is located on chromosome 3 but at a distance from the Adh-1/Adh-3 complex as previously reported. However, the new polymorphism identified in the 2.0-kb EcoRI fragment enabled this sequence to be mapped at the Adh-1/Adh-3 complex.