The ability of the tumor suppressor protein, p53, to recognize certain types of DNA lesions may represent one of the mechanisms by which this protein modulates cellular response to DNA damage. p53 DNA binding properties are regulated by several factors, such as post-translational modifications including phosphorylation and acetylation, regulation by its own C-terminal domain and interactions with other cellular proteins. Substrates resembling Holliday junctions and extra base bulges were used to study the effect of three nuclear proteins, HMG-1, HMG I(Y) and hMSH2–hMSH6, on the lesion binding properties of p53. Gel retardation assays revealed that the three proteins had varying effects on p53 binding to these substrates. HMG-1 did not influence p53 binding to Holliday junctions or 3-cytosine bulges. HMG I(Y) rapidly dissociated p53 complexes with Holliday junctions but not 3-cytosine bulges. Finally, the mismatch repair protein complex, hMSH2–hMSH6, enhanced p53 binding to both substrates by 3–4-fold. Together, these results demonstrate that p53 DNA binding activity is highly influenced by the presence of other proteins, some having a dominant effect while others have a negative effect.