We generated mice harboring germline mutations in which the enhancer element located 9 kb 3′ of the immunoglobulin κ light chain gene (3′Eκ) was replaced either by a single loxP site (3′EκΔ) or by a neomycin resistance gene (3′EκN). Mice homozygous for the 3′EκΔ mutation had substantially reduced numbers of κ-expressing B cells and increased numbers of λ-expressing B cells accompanied by decreased κ versus λ gene rearrangement. In these mutant mice, κ expression was reduced in resting B cells, but was normal in activated B cells. The homozygous 3′EκN mutation resulted in a similar but more pronounced phenotype. Both mutations acted in cis. These studies show that the 3′Eκ is critical for establishing the normal κ/λ ratio, but is not absolutely essential for κ gene rearrangement or, surprisingly, for normal κ expression in activated B cells. These studies also imply the existence of additional regulatory elements that have overlapping function with the 3′Eκ element.