Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein–protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (³¹⁴PAPV³¹⁷) of mouse IRF-2 to be different (³¹⁴SSSM³¹⁷) from human IRF-2. Recombinant GST-IRF-2 with ³¹⁴PAPV³¹⁷ (wild type) and ³¹⁴SSSM³¹⁷ (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with ³²P-(GAAAGT)₄ by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA–protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA–protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and influence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.