Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.