The regulation of interleukin (1L)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of IL-2. However, B cells can also synthesize IL-2. In the present study we analyzed the control of IL-2 promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting IL-2 at a low level after stimulation with phorbol 12myristate 13-acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the IL-2 promoter [distal nuclear factor (NF)-AT, proximal NF-AT, AP-l/Octamer (UPS) or NF-xB (TCEd) sites] were performed. In EBV-transformed B clones, the xB site exerted the strongest inducible activity; the NF-AT binding sites showed either no or only weak activity compared to Jurkat T cells. An IL-2 promoter bearing a defective NF-xB site was completely inactive in EBV-B cells, while it still had activity in Jurkat T cells. In seven EBV-B cell clones or lines differing in their capacity to secrete IL-2, the activity of the IL-2 promoter correlated well with the status of IL-2 secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through xB factors, was found to be active in the IL-2-producing EBV-B cells, but inactive in the non-IL-2-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the IL-2 NF-xB probe revealed the constitutive generation of xB complexes in IL-2-secreting cells consisting mainly of heterodimeric pSO/p6S complexes. A weaker xB complex formation and faster-migrating complexes were detected in non-IL-2-secreting cells. These results demonstrate that the IL-2 NF-xB site is indispensable for the activity of the IL-2 promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for IL-2 expression in these cells.