The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication. Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication. The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes. In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73alpha up-regulates the activity of this promoter. Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73alpha-induced p21 promoter activity. Using deletion mutants, we found that the DNA-binding domains of both p53 and p73alpha are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively. CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73alpha. These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance.