The t(14;18) chromosome translocation at the human bcl-2 gene locus causes bcl-2 deregulation, which inhibits programmed cell death and results in development of human follicular B-cell lymphoma. Transcriptionally active genes are typically embedded in DNase I-sensitive “domains” extending many kilobases to either side, consistent with the notion that decondensation of chromatin increases the accessibility of cis-acting elements to trans-acting factors. I used DNase I to examine the possible roles played by the alterations of chromatin structures in t(14;18) translocation and/or bcl-2 gene deregulation. Nuclei were isolated from cultured lymphocytes and digested with DNase I at low concentrations. Purified DNA was subjected to Southern analysis with unique probes isolated from the third intron of the bcl-2 gene. Several DNase I hypersensitive sites were detected near the translocation breakpoints. One site was mapped to a region in the third intron and detectable in cells without the t(14;18) translocation, such as DHL 2 and DHL 9 cells. Furthermore, the sequence of one DNase I hypersensitive site was found to closely resemble a promoter. Northern blot analysis and RNase protection assays using this putative promoter sequence as a probe detected a RNA species in certain fetal tissues, especially brain. The demonstration of DNase I hypersensitivity and gene activity at the t(14;18) breakpoints is important for understanding the structural organization of the bcl-2 locus, its gene regulation in normal cells, and deregulation in follicular B-cell lymphoma.