Adult T-cell leukaemia (ATL)-derived factor (ADF), originally described as an inducer of interleukin-2 receptor-alpha (IL-2R alpha/Tac), has homology with the co-enzyme thioredoxin which is involved in many dithiol-dependent reducing processes. Using antibody against the C-terminal synthetic polypeptide of ADF and RNA probe of ADF, we examined the expression of ADF in various cell lines by immunofluorescence, immunohistochemical staining, Western blotting and in situ hybridization. ADF was intensely expressed on HTLV-I+ T-cell lines as compared with HTLV-I- T-cell lines. While the Epstein-Barr virus (EBV)+ B-lymphoblastoid cell lines were intensely positive for ADF, Burkitt-derived B cell line Jijoye with defective EBV was negative for ADF. Electron microscopic and photomicroscopic analysis of HTLV-I+ ATL-2 cells showed that ADF was localized on both the cell membrane and cytosol. In ATL-2 cells, a marked heterogeneity of ADF expression was observed. In in situ hybridization, heterogeneity of ADF messenger RNA (mRNA) expression was also demonstrated, indicating that ADF expression was regulated in the transcriptional level.