Spi-B and PU.1 are hematopoietic-specific transcription factors that constitute a subfamily of the Ets family of DNA-binding proteins. Here we show that contrary to previous reports, PU.1 and Spi-B have very different expression patterns. PU.1 is expressed at high levels in B cells, mast cells, megakaryocytes, macrophages, neutrophils, and immature erythroid cells and at lower levels in mature erythrocytes. PU.1 is completely absent from peripheral T cells and most T cell lines based on sensitive RT-PCR assays. In contrast, Spi-B is expressed exclusively in lymphoid cells and can be detected in early fetal thymus and spleen. In situ hybridizations of adult murine tissues demonstrate Spi-B mRNA in the medulla of the thymus, the white pulp of the spleen, and the germinal centers of lymph nodes. Spi-B expression is very abundant in B cells and both Spi-B mRNA and protein are detected in some T cells. In situ hybridization and Northern blot analysis suggest that Spi-B gene expression increases during B cell maturation and decreases during T cell maturation. Gel-retardation experiments show that Spi-B can bind to all putative PU.1 binding sites, but do not reveal any preferred Spi-B binding site. Finally, both PU.1 and Spi-B function as transcriptional activators of the immunoglobulin light-chain enhancer E lambda 2.4 when coexpressed with Pip (PU.1-interaction partner) in NIH-3T3 cells. Taken together, these data suggest that differences in patterns of expression between Spi-B and PU.1 distinguish the function of each protein during development of the immune system.