Murine B-lineage cells can express either kappa or lambda Ig light chains. However, most of these cells express kappa protein, a phenomenon that appears to be controlled in part at the level of gene rearrangement. This feature may reflect a preference for the kappa locus by the recombinase machinery, or it may indicate that kappa rearrangement begins before lambda rearrangement. These possibilities can be distinguished by measuring the kinetics with which light chain gene rearrangement at both loci actually occurs. To this end, we have used pre-B cells transformed by temperature-sensitive mutants of Abelson virus that undergo kappa and lambda gene rearrangement when shifted to the nonpermissive temperature. Competitive PCR analyses of rearrangement kinetics demonstrate that the kappa and lambda loci rearrange at about the same time in these cells. Consistent with this, some clones isolated from cells induced for a short period of time have rearranged only the lambda locus. However, the frequency with which lambda genes are rearranged is three- to sixfold lower than that with which kappa genes rearrange. These data indicate that the recombinase machinery targets both light chain loci at the same time, but acts preferentially at the kappa locus. The reduced ability of the recombinase machinery to target the lambda locus and selection pressures occurring during B cell development probably both contribute to the preferential usage of kappa genes in normal murine B lineage cells.