The binding of the Ku protein to DNA was investigated using the atomic force microscope. Ku was found to bind predominantly to the ends of double-stranded DNA. Experiments with plasmid DNA revealed that Ku does not bind to circular plasmids but does bind to plasmids that have been linearized by treatment with ionizing radiation. The binding of Ku to poly(dG-dC) x poly(dG-dC) polynucleotides and to a 400-bp DNA EcoRI fragment resulted in a shift in the fragment size distribution to include longer fragments, with internally binding Ku. Furthermore, we observed images consistent with fragments joined together by Ku, showing an interaction with two ends of DNA. These observations suggest that Ku may play a role in physically orienting DNA for ligation by binding the ends of adjacent DNA molecules.