As a first step toward defining the elements necessary for λ immunoglobulin gene regulation, DNase I hypersensitive sites were mapped in the mouse λ locus. A hypersensitive site found 15.5 kb downstream of Cλ4 was present in all the B-cell but not in the T-cell lines tested. This site coincided with a strong B-cell-specific transcriptional enhancer (E(λ2-4)). This novel enhancer is active in myeloma cells, regardless of the status of endogenous λ genes, but is inactive in a T-cell line and in fibroblasts. The enhancer E(λ2-4) functions in the absence of the transcription factor NFκB, which is necessary for κ enhancer function. No evidence could be found for NFκB binding by this element. Rearrangement of Vλ2 to JCλ3 or JCλ genes deletes E(λ2-4); however, a second strong enhancer was found 35 kb downstream of Cλ1, which cannot be eliminated by λ gene rearrangements. The second λ enhancer (E(λ3-1)) is 90% homologous to the E(λ2-4) sequence in the region determined to comprise the active enhancer and likewise lacks the consensus binding site for NFκB. The data support a model for the independent activation of κ and λ gene expression based on locus-specific regulation at the enhancer level.