Abstract The use of 129 strain-derived embryonic stem cell lines for targeted gene mutation in mice has led directly to an expanded use of this inbred strain worldwide. It has been noted, however, that the 129 genetic background can make a significant contribution to the severity of a mutant phenotype. In this study, we reveal a specific defect in the IL-5 and Rp105 responses of B lymphocytes from two widely used 129 mouse substrains. The response to stimulation through surface IgM is also diminished, although to a lesser degree, in these mice. The lesion appears to reduce significantly the expression of the α-chain of the IL-5R, but may also influence events downstream of the IL-5R. This phenotype displays a codominant inheritance pattern, and is accompanied by a variable but significant depression of peritoneal B-1 cell numbers in 50% of the mice.