We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.l putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-l/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19ql3.3-19ql3.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.