Previous work has indicated that inbred mouse strains C57BL/6 and DBA/2 produce red cells differing in their sensitivity to osmotic lysis and that the trait is under multigene control. A recombinant inbred strain (BXD-31), produced from C57BL/6 and DBA/2, has red cells manifesting resistance to osmotic lysis far greater than that of either progenitor. We demonstrate here that the fragility difference between BXD-31 and DBA/2 is the consequence of allelic variation at a single autosomal locus, termed rol. The resistance allele (rol(r)) is almost completely recessive to the sensitive one (rol(s)). Results of bone marrow chimera analyses indicate that (1) the mode of rol gene action is by a direct influence on the properties of the red cells rather than an indirect influence on their extracellular milieu, and (2) rol does not affect erythrocyte production and turnover. The fragility difference caused by rol variation is likely to involve the erythrocyte membrane or underlying cytoskeleton, since various red cell properties sensitive to ion metabolism differences are unaffected by the gene.