Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat–binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3′ overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3′ telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.