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Adult T-cell leukemia/lymphoma (ATLL) is a T-cell malignancy associated with HTLV-1. The HTLV-1 provirus genome has the pX region that encodes tax and HTLV-1 basic leucine zipper factor (HBZ). Previous studies have reported that the tax gene is expressed in few ATLL cases, but the HBZ gene in all ATLL cases. In this study, we used HBZ gene in situ hybridization (HBZ-ISH) for detection of the HBZ gene in formalin-fixed paraffin-embedded tissues. This method showed that all cases (n = 19) were positive for the ATLL cell line (MT-1, MT-2, and MT-4) and ATLL mouse model (HBZ-Tg mice and NOD/SCID/β2-microglobulinnull mice with ATLL transplanted), and the HBZ gene was also detected in all human ATLL cases (n = 16). The percentage of positive cells in HBZ-ISH was 5–70%. Immunohistochemical staining for Tax protein showed positivity in seven of 11 cases in NOD/SCID/β2-microglobulinnull mice with ATLL transplanted and in six of eight human ATLL cases, but the percentage of positive cells was very low (range, 1–5%). Although HBZ-ISH is unsuitable to detect HTLV-1 clonality, this method is convenient and can be useful for the histological diagnosis of ATLL in HTLV-1 sero-indeterminate patients. (Cancer Sci 2011; 102: 1432–1436)