The yeast TBF1 gene is essential for mitotic growth and encodes a protein that binds the human telomere repeats in vitro, although its cellular function is unknown. The sequence of the DNA-binding domain of Tbf1p is more closely related to that of the human telomeric proteins TRF1 and TRF2 than to any yeast protein sequence, yet the functional homologue of TRF1 and TRF2 is thought to be Rap1p. In this study we show that the Tbf1p DNA-binding domain can target the Gal4 transactivation domain to a (TTAGGG)(n) sequence inserted in the yeast genome, supporting the model that Tbf1p binds this sub-telomeric repeat motif in vivo. Immunofluorescence of Tbf1p shows a spotty pattern throughout the interphase nucleus and along synapsed chromosomes in meiosis, suggesting that Tbf1p binds internal chromosomal sites in addition to sub-telomeric regions. PCR-assisted binding site selection was used to define a consensus for high affinity Tbf1p-binding sites. Compilation of 50 selected oligonucleotides identified the consensus TAGGGTTGG. Five potential Tbf1p-binding sites resulting from a search of the total yeast genome were tested directly in gel shift assays and shown to bind Tbf1p efficiently in vitro, thus confirming this as a valid consensus for Tbf1p recognition.