Ku is a heterodimeric protein involved in nonhomologous end-joining of the DNA double-stranded break repair pathway. It binds to the double-stranded DNA ends and then activates a series of repair enzymes that join the broken DNA. In addition to its function in DNA repair, the yeast Saccharomyces cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes that affect telomere function. The yeast telomeres are composed of duplex C1-3(A/T)G1-3 telomeric DNA repeats plus single-stranded TG1-3 telomeric DNA tails. Here we show that Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that mimics the structure of telomeres. Addition of Cdc13p, a single-stranded telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of a ternary complex with Cdc13p binding to the single-stranded tail of the DNA substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail inhibits the binding of Yku, the results suggested that loading of Yku and Cdc13p to telomeres is sequential. Through generating a double-stranded break near telomeric DNA sequences, we found that Ku protein appears to bind to the de novo synthesized telomeres earlier than that of Cdc13p in vivo. Thus, our results indicated that Yku interacts directly with telomeres and that sequential loading of Yku followed by Cdc13p to telomeres is required for both proteins to form a ternary complex on telomeres. Our results also offer a mechanism that the binding of Cdc13p to telomeres might prevent Yku from initiating DNA double-stranded break repair pathway on telomeres.