Recognition of certain types of DNA lesions by the tumor suppressor protein, p53, represents one of the several downstream functions of this protein in response to DNA damage. This binding property is regulated by several factors including posttranslational modifications and interactions with other proteins. Phosphorylation by several stress-response kinases activates p53 by increasing protein stability as well as transactivation properties. Here we examined the effect of phosphorylation events on the sequence-independent binding properties of p53 using two DNA substrates: One resembling Holliday junctions and the other containing extra base bulges. Gel retardation assays showed that dephosphorylation of serine 392 in the C-terminal domain of p53 greatly reduces Holliday junction and lesion recognition. In contrast, sequence-specific binding is disrupted by the removal of some N-terminal phosphates but not serine 392. Rephosphorylation of p53 by certain kinases can restore p53 recognition of Holliday junctions and 3-cytosine bulges. In all cases, phosphorylation of serine 392 occurs; however, reactivation also involves other residues. Together, the results show that p53 DNA binding activity is strongly regulated by the phosphorylation state of the protein.