The V_H repertoire on both H chain alleles of normal and λ5-deficient B lineage cells were analyzed by single-cell PCR. The μH chains were tested for their capacity to form a pre-B cell receptor. In bone marrow, D-proximal V_H genes were found preferentially expressed in λ5-deficient pre-B cells and in a newly identified early c-kit⁺ cytoplasmic μH chain⁺ pre-B cell population of normal mice. Only half of the μH chains expressed in these cells have the capacity to form a pre-B cell receptor. Representation of the D-proximal V_H genes was found suppressed on the productive but not on the nonproductive V_HDJ_H rearranged alleles of c-kit⁻ preB-II cells and splenic λ5-deficient B cells. More than 95% of the μH chains expressed in preB-II cells can form a pre-B cell receptor. These results demonstrate that the pre-B cell receptor in normal mice and the B cell receptor in λ5-deficient mice mediate a shift in the V_H repertoire.