We initially identified c-myc promoter-binding protein 1 (MBP-1) from a human cervical carcinoma cell expression library which negatively regulates c-myc promoter activity. A recent study demonstrated that MBP-1 acts as a general transcriptional repressor (A. K. Ghosh, R. Steele, and R. B. Ray, Mol. Cell. Biol. 19:2880-2886, 1999). In order to identify the cellular protein(s) interacting with MBP-1 for transcriptional regulation, a HeLa cell cDNA expression library was screened using a yeast two-hybrid system. An MBP-1-interacting cDNA encoding a polypeptide of 140 amino acid residues with an approximate molecular mass of 16 kDa was identified and named MBP-1 interacting protein-2A (MIP-2A). MIP-2A has a sequence similarity with an unknown mRNA and SEDL. Mutations in the SEDL gene, located at human chromosome Xp22, has recently been implicated with an X-linked genetic disease, although the function of SEDL gene product was not determined (A. K. Gedeon et al., Nat. Genet. 22:400-404, 1999). However, our results suggested the localization of MIP-2A at human chromosome 19. The specificity of interaction between MBP-1 and MIP-2A was verified by an in vitro glutathione S-transferase pulldown experiment, a mammalian two-hybrid analysis, and in vivo coimmunoprecipitation assays. Further analysis revealed that the amino-terminal domain of MBP-1 (amino acids 1 to 95) interacts with MIP-2A. Immunofluorescent staining suggested colocalization of MIP-2A and MBP-1 primarily in the perinuclear membrane of cells. Functional analysis demonstrated that MIP-2A relieves MBP-1 mediated transcriptional repression on c-myc promoter. Additionally, MIP-2A antagonizes cell growth regulatory role of MBP-1. Taken together, these results suggest the functional interaction of MIP-2A and MBP-1 in cell growth regulation.