The Shelterin complex associates with telomeres and plays an essential role in telomere protection and telomerase regulation. In its most abundant form, the complex is composed of six core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Of these subunits, three can interact directly with either single-stranded (POT1) or double-stranded (TRF1, TRF2) telomeric DNA. In this report, we have developed assays to measure the DNA binding activity of Shelterin complexes in human cell extracts. With these assays, we have characterized the composition and DNA binding specificity of two Shelterin complexes: a 6-member complex that contains all six core components and a second complex that lacks TRF1. Our results show that both of these complexes bind with high affinity (K(D) = 1.3-1.5 × 10(-9) M) and selectively to ds/ss-DNA junctions that carry both a binding site for POT1 (ss-TTAGGGTTAG) and a binding site for the SANT/Myb domain of TRF1 or TRF2 (ds-TTAGGGTTA). This DNA binding specificity suggests the preferential recruitment of these complexes to areas of the telomere where ss- and ds-DNA are in close proximity, such as the 3'-telomeric overhang, telomeric DNA bubbles and the D-loop at the base of T-loops.