Stage-specific activator protein (SSAP) is the transcription factor responsible for the activation of the sea urchin late H1 gene at the mid-blastula stage of embryogenesis. SSAP contains an extremely potent transcription activation domain that functions 4-5-fold better than VP16 in a variety of mammalian cell lines. We used the two-hybrid screening technique to identify human cDNAs from an HL60 cell-derived cDNA library that encode proteins that interact with the transcription activation domain of SSAP. One of these cDNAs encodes ZFM1, a protein previously identified at the locus linked to multiple endocrine neoplasia type 1 (MEN1) and as presplicing factor SF1. Functional assays establish the ZFM1 protein as a transcriptional repressor. ZFM1 protein represses Gal4-GQC-mediated transcription, and this activity requires both a repression domain found in the N-terminal 137 amino acids of the protein, as well as a GQC interaction region. The physiological significance of repression mediated by ZFM1 comes from the ability of its specific repression domain to function when fused to Gal4 and tethered to promoters containing Gal4 binding sites. The activity is unique in that activated but not basal transcription levels are affected.