The human adenine nucleotide translocase‐2 (ANT2) promoter contains a silencer region that confers partial repression on the heterologous herpes simplex virus thymidine kinase (HSVtk) promoter [Barath, P., Albert‐Fournier, B., Luciakova, K., Nelson, B.D. (1999) J. Biol. Chem.274, 3378–3384]. Two sequences in the silencer (Site‐2 and Site‐3) are protected in the DNase I assay in vitro, and one of these is a repeated GTCCTG element previously shown to act as the active repressor element. We have now purified the DNA binding protein, and identified it using MALDI‐TOF MS as a 33‐kDa member of the nuclear factor 1 (NF1) family of transcription factors. NF1 purified from rat liver and HeLa cell nuclei bind to both silencer Site‐2 and Site‐3, resulting in a DNase I footprint identical to that obtained with purified recombinant NF1. Furthermore, transient transfection experiments with reporter constructs containing mutated silencer Site‐2 and/or Site‐3 show that both sites contribute to repression of the HSVtk promoter. Finally, chromatin immunoprecipitation analysis reveals that NF1 is bound to both elements on the endogenous HeLa cell ANT2 promoter. Our data support the belief that NF1 acts as a repressor when bound to silencing Site‐2 and Site‐3 of the ANT2 gene.