We have isolated a cDNA encoding a transcription factor that binds to the enhancer of major histocompatibility complex (MHC) class I genes. MHC-binding protein 2 (MBP-2) is a 275-kDa protein, containing two sets of widely separated zinc fingers and a stretch of highly acidic amino acids, a putative transactivation domain. The two zinc finger regions, when expressed individually as bacterial fusion proteins, bind with highest affinity to the MHC class I gene enhancer. Several proteins found in mammalian nuclear extracts bind the MHC class I enhancer in an electrophoresis mobility shift assay. Only one of these, a ubiquitously expressed factor, forming a slow-migrating retarded complex, can be supershifted by a MBP-2 antiserum. The same antiserum also precipitates a protein of greater than 250 kDa from COS cells transfected with a MBP-2 expression vector. Our data indicate that MBP-2 is a transcription factor involved in the regulation of MHC class I gene expression.