The recombination activating genes (RAGs) play a critical role in V(D)J recombination machinery and lymphocyte development. Their expression is strictly regulated during lymphocyte ontogeny, with expression being rapidly lost as the lymphoid precursors differentiate into their progeny. To elucidate molecular mechanisms of regulation of human RAG-1 gene expression, we examined a chromatin structure of a approximately 24-kb DNA segment adjacent to a human RAG-1 promoter region in various cell lines by analyzing DNase I hypersensitive (DNase I HS) sites. In a RAG-1-expressing human pre-B-cell line, at least four DNase I HS sites (HS1, HS2, HS3, and HS4) were identified. Among these HS sites, one HS site (HS1) was ubiquitously detected in all cell lines examined, but the other three HS sites (HS2, HS3, and HS4) were associated only with RAG-1-expressing lymphoid cell lines. Using transient expression assays, we showed that the 5' upstream region of the major transcription start site showed low but significant promoter activity and that a DNA segment within HS3 located in the promoter region was indispensable to its basal promoter activity. Importantly, this promoter region was shown to be active in both RAG-1-expressing and RAG-1-nonexpressing cell lines. These results suggest that alteration of chromatin structure in the promoter region, in addition to other control elements outside of the promoter region, is one of the mechanisms regulating tissue- and stage-specific expression of human RAG-1 gene.