The mammalian transcription factor, NF-Y(CBF), contains three known subunit components, NF-YA (CBF-B), NF-YB(CBF-A), and NF-YC(CBF-C), which are all required to reconstitute specific CCAAT box DNA binding activity. In this study, the high mobility group chromosomal protein, HMG-I(Y), has been shown to activate NF-Y in transient transfections in vivo using the natural murine alpha2(I) collagen promoter and a multimerized version of the proximal NF-Y(CBF) CCAAT box element. In vitro analysis of the alpha2(I) collagen promoter region inclusive of the NF-Y(CBF) binding site (-106 to -65 base pairs) failed to identify any high affinity HMG-I(Y) DNA-binding sites. However, the heterotrimeric NF-Y complex, as well as the NF-YA subunit alone, was shown to stably interact in vitro with both HMG-I(Y) and phosphorylated HMG-I, as modified by casein kinase II, using far Western and protein-protein interaction solution assays in the absence of CCAAT box DNA. Furthermore, the interaction between HMG-I(Y) and NF-Y was mapped to the highly conserved DNA binding-subunit interaction domain (DBD) of the NF-YA subunit and to a single AT-hook motif in HMG-I(Y). Recombinant HMG-I was also found to stabilize the CCAAT box DNA binding activity of recombinant NF-Y, as well as the native NF-Y complex, in vitro. Together, these results suggest a functional HMG-I(Y) protein binding site has been identified in the NF-Y complex and mapped to the conserved DBD and AT-hook regions of NF-YA and HMG-I(Y), respectively. This protein-protein interaction site may function to modulate NF-Y activity through stabilization of NF-Y binding to its CCAAT box DNA-binding site.