Many inbred strains of mice have a single locus encoding renin, Ren-1, whereas other inbred strains have two tandemly linked loci, Ren-1 and Ren-2. Each of these renin genes in inbred mice exhibits a unique pattern of tissue-specific expression. As a prerequisite to understanding the structural basis for the expression differences, we have physically characterized the sequence organization of this chromosomal region in both types of strains. Pulsed field gel electrophoresis was initially used to compare the long-range structure of this region in C57BL/6 (Ren-1) and DBA/2 (Ren-1 + Ren-2) mice. The structure in both inbred strains is extremely similar, except for an additional 30 kb containing Ren-2 in DBA/2 mice. The boundaries of the extra 30-kb segment were sequenced and compared to homologous sequences flanking the Ren-1 alleles. This analysis identified the precise recombination site, and also the presence of a large insertion, between the renin loci in DBA/2. The renin gene duplication apparently resulted from recombination between sequences sharing little homology, suggesting that nonhomologous chromosomal breakage and rejoining may have been involved mechanistically in the event.