V(D)J rearrangement, high level expression and somatic hypermutation of assembled Ig genes is tightly controlled by a number of regulatory sequence elements located in the vicinity of the J-, (D)-, and C-gene segments. During B cell maturation these elements become accessible to binding of trans-acting factors, reflecting the opening of the chromatin structure through an as yet unidentified mechanism. The mapping of regions of an altered chromatin structure (DNase I hypersensitivity) therefore is a powerful approach in identifying regulatory sequence elements. We here show that the human Ig lambda enhancer consists of three modules previously identified by us as DNase I-hypersensitive sites HSS-1, -2, and -3. The three sequence elements synergize in transcriptional activation of a reporter gene and together constitute a powerful tissue-specific enhancer which is a much stronger transcriptional activator than the kappa enhancers alone or in combination. We further show that the accessibility of the kappa and lambda enhancer elements for DNase I in the chromatin of a pre-B cell line (207) correlates with the transcriptional enhancer activities of kappa and lambda enhancer constructs. This finding is in support of an ordered model for Ig light chain activation (kappa before lambda).