We have previously described purification and characterization of a nuclear protein, TREF, which interacts specifically with the transcriptional control element, TRA, of the human transferrin receptor (TR) gene. In this report we show that TREF can be separated into two functionally distinct DNA-binding activities. The first DNA-binding activity (TRAC) is highly specific for the 8-bp element TRA and the related Escherichia coli cAMP receptor binding site. This motif is homologous to the phorbol 12-tetradecanoate 13-acetate- and cAMP-responsive elements of eukaryotic genes and the regulatory proximal sequence elements of the U1 small nuclear RNA gene and is also present in the promoter of the Drosophila melanogaster yolk protein factor 1 gene. In striking contrast, the second activity exhibits high affinity for the ends of double-stranded DNA in a sequence-unspecific manner and is attributable to the heterodimeric Ku autoantigen. Notably, transcription of Ku is induced during mid-late G0/G1 with kinetics similar to the TR gene. Ku is a highly abundant nuclear protein possessing nonspecific affinity for the ends of DNA, whose biological role remains to be elucidated. A transcriptional role for this protein has been proposed, however, on the basis of studies attributing DNA sequence-specific binding activity, notably for TRA-like sequences described above, directly to the Ku heterodimer. The observation that Ku-mediated nonspecific DNA-binding activity copurifies with the TRA-specific activity, TRAC, clearly has implications for these and related studies. The unusual properties of TRAC activity and its relationship, if any, with the enigmatic Ku protein, are discussed.