Spi-B and PU.1 (Spi-1) comprise the most divergent subfamily of the Ets transcription factor family. Spi-B and PU.1 bind to similar DNA sequences, and can activate the same B-cell and myeloid promoters in vitro. However, PU.1 knockout mice demonstrate defective hematopoietic development of multiple hematopoietic lineages, indicating that Spi-B was not able to compensate for loss of PU.1. One explanation for these results is that, in contrast to PU.1, which is expressed in myeloid and B-cell lines, Spi-B expression is restricted to B-cells. In order to begin to understand the control of regulation of the Spi-B gene, murine Spi-B cDNA and genomic clones were isolated. The exon/intron organization and transcriptional start sites were determined; two major transcriptional start sites were detected. The two Spi-B promoters were isolated and characterized, and displayed differential activity in B-cell lines matching that of the endogenous gene. Further study of the two Spi-B promoters will provide insight into the molecular events regulating the tissue-specific and developmental stage-specific expression of Spi-B in B-cells.