A novel human gene, LAF-4, was isolated from a subtracted cDNA library that showed strong sequence similarity to AF-4, a gene that is translocated in t(4;11)(q21;q23) acute lymphoblastic leukemias (ALLs). In t(4;11) ALL, the AF-4 gene at 4q21 is translocated into the MLL locus at 11q23, resulting in the expression of an MLL/AF-4 fusion protein that is the presumptive oncoprotein. AF-4 and LAF-4 are homologous throughout their coding regions, yet neither protein is related to previously cloned genes. Human LAF-4 readily hybridized with genes in mouse and chicken, thus showing that this gene family has been highly conserved during vertebrate evolution. In mouse tissues, LAF-4 mRNA was found to be present at highest levels in lymphoid tissues, present at lower levels in brain and lung, and absent from other tissues. In human and mouse lymphoid cell lines, LAF-4 expression was highest in pre-B cells, intermediate in mature B cells, and absent in plasma cells, thus pointing to a potential regulatory role for LAF-4 in lymphoid development. Antibodies to LAF-4 showed it to be a nuclear protein that showed an uneven, granular immunofluorescence pattern. In vitro-translated LAF-4 was able to bind strongly to double-stranded DNA cellulose. Furthermore, both LAF-4 and AF-4 had domains that activated transcription strongly when fused to the GAL4 DNA-binding domain. Interestingly, the AF-4 transactivation domain is retained in the MLL/AF-4 fusion protein; thus, it may contribute to the transforming potential of the oncoprotein. Therefore, the cloning of LAF-4 has defined a new family of potential regulatory proteins that may function in lymphoid development and oncogenesis.