The CDC13 gene of Saccharomyces cerevisiae is required both to protect telomeric DNA and to ensure proper function of yeast telomerase in vivo. We have previously demonstrated that Cdc13p has a high affinity single-strand telomeric DNA binding activity, although the primary amino acid sequence of Cdc13p has no previously characterized DNA binding motifs. We report here mapping of the Cdc13 DNA binding domain by a combination of proteolysis mapping and deletion cloning. The DNA binding domain maps to residues 557–694 of the 924-amino acid Cdc13 polypeptide, within the most basic region of Cdc13p. A slightly larger version of this domain can be efficiently expressed in Escherichia coli as a soluble small protein, with DNA binding properties comparable to those of the full-length protein. A single amino acid missense mutation within this domain results in thermolabile DNA binding and conditional lethality in yeast, consistent with the prediction that DNA binding should be essential for CDC13 function. These results show that Cdc13p contains a discrete substructure responsible for DNA binding and should facilitate structural characterization of this telomere binding protein.