Using a subtractive cDNA approach we have identified two nearly identical genes, Xlr3a and Xlr3b (X-linked lymphocyte regulated), expressed at a consistently high level in 14 out of 14 murine plasmacytoma cell lines, at a high level in 1 out of 8 B-lymphoma cell lines, and at a very low level in 2 out of the 8 B-lymphoma cell lines. The messages are not detected in 10 pre-B-lymphoma cell lines. These genes express 2.0-kb mRNAs that encode 226-amino-acid proteins that are extremely basic, with an estimated pI of 8.1 and 9.0, respectively. By sequence comparison they are homologous to Xlr1, an acidic nuclear protein that is produced in lymphoid cell lines corresponding to the late stages of lymphocyte differentiation. Xlr2 is a highly homologous gene that is expressed in differentiating male germ cells. Xlr3a and Xlr3b are members of a new subfamily in the Xlr multigene family. Like Xlr1, they are up-regulated during B-cell terminal differentiation in normal and neoplastic B-cells, and cross-hybridize with a message in testis RNA. Also, like Xlr1, they do not cross-hybridize with human genomic DNA.