Nuclear extracts from maize leaves have been shown previously to contain a factor, MNF1, that interacts with both the cauliflower mosaic virus 35S promoter and the promoter of the maize gene for phosphoenolpyruvate carboxylase, which is involved in C4 photosynthesis. We have isolated two cDNA clones encoding proteins (MNB1a and MNB1b), that bind to an MNF1-binding site in a sequence-specific manner, by screening of a maize cDNA expression library with synthetic oligonucleotides. Using various mutated oligonucleotides, we showed that both proteins recognize an AAGG motif at the MNF1-binding site as important bases for binding, as does MNF1 in maize nuclear extracts. However, the binding specificities of MNB1a and MNB1b are similar but not identical to that of MNF1. The deduced amino acid sequences of these proteins are completely different from each other. The basic region of MNB1b exhibits homology to the high mobility group (HMG) box of the vertebrate HMG1 family, whereas MNB1a exhibits no homology to any known proteins. Southern blot analysis of genomic DNA revealed that the cDNA for MNB1a is derived from a multigene family whose members have highly homologous N-terminal basic domain, whereas the gene for MNB1b exhibits only limited homology to a few other genes. These results suggest that the MNF1-binding site on the 35S promoter is a target of multiple DNA-binding proteins.