Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73beta proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73beta than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73beta but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73beta than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Delta30) demonstrates the same sequence discrimination as does p73beta. In vivo, both p53 and p73beta exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites.