Resistance to radiation leukemia virus-induced leukemia is mediated by gene(s) in the H-2D region of the MHC; a clear correlation exists between disease resistance and increased H-2Dd expression on the thymocyte surface. We have investigated the molecular basis for this stimulation of H-2Dd class I expression. Elevated H-2 mRNA and H-2 transcription are demonstrated in the infected thymocytes as compared to normal thymocytes indicating that the elevation of H-2 surface expression is the result of transcriptional activation. Gel mobility assays performed with nuclear extracts of normal and infected thymocytes and sequences 5' of the H-2Dd gene show that specific binding occurs with both extracts; the binding differs both quantitatively and qualitatively, however. DNase I protection analysis detects a protein binding site that is protected only by extracts from infected cells. The protected region contains a sequence similar to the AP-1 consensus sequence. Gel shift competition assays and UV photo-cross-linking to an oligonucleotide containing this sequence demonstrate that specific binding of an H-2 binding factor 1 occurs and that this factor is not the AP-1 binding complex. This novel binding factor, activated in vivo, might also be involved in the normal regulation of H-2 gene expression by recognizing the highly conserved binding sequence (TGACGCG) found in the 5' flanking region of many MHC class I genes. This is the first demonstration of the parallel stimulation of a DNA binding activity and increased transcription occurring in thymocytes after infection with a leukemogenic retrovirus.