The class II (Ia) major histocompatibility complex antigens are a family of integral membrane proteins whose expression is limited to certain cell types, predominantly B lymphocytes, macrophages, and thymic epithelial cells. In B cells, Ia expression is both developmentally regulated and responsive to external stimuli. The differentiation of early B stem cells to mature B lymphocytes is accompanied by the appearance of cell surface Ia antigens; the transition to plasma cells results in loss of class II gene expression. In Ia-expressing B cells, the T cell-derived lymphokine interleukin-4 (IL-4) increases such expression by an as yet undefined mechanism. Chloramphenicol acetyltransferase gene expression was cis-activated by a region of the Ia A alpha k gene in a B lymphoma line, but not in a myeloma line. A nuclear protein that bound to two sites within this region, upstream from previously described transcription elements, was found in normal spleen cells. This binding activity was also found in spleen extracts from athymic mice, which lack T lymphocytes, and in Ia-positive B lymphocyte tumor cell lines, demonstrating that it is a B cell protein. Further analysis showed the activity to be undetectable in an Ia-negative pre-B cell line and in three plasmacytoma cell lines that are Ia negative. IL-4 treatment of normal and athymic mouse spleen cells greatly increased the binding of this nuclear protein to these two sites, concomitant with increased MHC class II gene transcription. Thus, B cells contain a sequence-specific DNA-binding activity whose level is influenced both by IL-4 and by differentiation signals.