The interaction of p53 with a human model telomere in vitro was examined by electron microscopy. p53 demonstrated a sequence-independent affinity for telomeric DNA in vitro, localizing to the 3' single strand overhang and the t-loop junction both in the presence and absence of associated TRF2. Binding was not observed above background along the duplex telomeric repeats. However, the efficiency of TRF2-catalyzed t-loop formation on the model DNA was increased 2-fold in the presence of p53 although a variety of single strand or Holliday junction-binding proteins did not facilitate t-loop formation. These results suggest that p53 has an active role in telomere maintenance and structure through association with the t-loop junction.