We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.