As a first step toward defining the elements necessary for lambda immunoglobulin gene regulation, DNase I hypersensitive sites were mapped in the mouse lambda locus. A hypersensitive site found 15.5 kb downstream of C lambda 4 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 lambda 2-4). This novel enhancer is active in myeloma cells, regardless of the status of endogenous lambda genes, but is inactive in a T-cell line and in fibroblasts. The enhancer E lambda 2-4 functions in the absence of the transcription factor NF kappa B, which is necessary for kappa enhancer function. No evidence could be found for NF kappa B binding by this element. Rearrangement of V lambda 2 to JC lambda 3 or JC lambda genes deletes E lambda 2-4; however, a second strong enhancer was found 35 kb downstream of C lambda 1, which cannot be eliminated by lambda gene rearrangements. The second lambda enhancer (E lambda 3-1) is 90% homologous to the E lambda 2-4 sequence in the region determined to comprise the active enhancer and likewise lacks the consensus binding site for NF kappa B. The data support a model for the independent activation of kappa and lambda gene expression based on locus-specific regulation at the enhancer level.