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The class II (Ia) major histocompatibility complex (MHC) antigens are a family of integral membrane proteins whose expression is limited to certain cell types. A pair of consensus sequences, X and Y, is found upstream of all class II genes, and deletion of each of these sequences eliminates expression of transfected genes. Furthermore, the absence of a specific X box binding protein in patients with severe combined immunodeficiency disease whose cells lack class II suggests an important role for these proteins in class II regulation. Here, the cloning of two lambda gt11 complementary DNAs encoding DNA binding proteins (murine X box binding proteins lambda mXBP and lambda mXBP-2) is reported. Both phage-encoded fusion proteins bind specifically to the X box of the A alpha, but not to E alpha or E beta class II genes. These two independent isolates do not cross-hybridize. The lambda mXBP complementary DNA hybridizes to two RNA species, 6.2 and 3.0 kilobases in mouse, that are expressed in both Ia positive and Ia negative cells. By means of DNA blot analysis with the lambda mXBP complementary DNA insert and probes generated from each end of this complementary DNA insert, lambda mXBP was found to arise from a multigene family. These data illustrate the high degree of complexity in the transcriptional control of this coordinately regulated gene family.