We used an electrophoretic mobility-shift assay to study proteins that bind to sequences in the 5' flanking region of the murine c-myc gene. By comparing the DNA-protein complexes formed with extracts from cells representing earlier stages of B-cell development with those from plasmacytomas, we identified a plasmacytoma-specific protein that binds to a region within the c-myc promoter. Five other regions of this promoter show extensive sequence-specific binding, but the binding is not clearly B-cell stage-specific. Methylation-interference and o-phenanthroline/copper-protection experiments identified a single plasmacytoma-specific protein binding site 290 base pairs 5' of the transcription start site P1. Homologues of a core sequence, d(AGAAAGGGAAAGGA), within the 25-base-pair binding site are found at three additional sites in the murine c-myc locus. The plasmacytoma-specific occurrence of this protein suggests that it may play a role in the transcriptional repression of the normal c-myc gene observed in plasmacytomas.