Native DNA from four strains of herpes simplex virus 1 (HSV-1) circularized after digestion with the lambda exonuclease, indicating that the molecules were terminally repetitious. In two strains, the terminal repetition was evident in nearly 50% of the DNA molecules. Maximal circularization was observed when only 0.25 to 0.5% of the DNA was depolymerized by the exonuclease, suggesting that the minimal size of the terminally repetitious regions is in the range of 400 to 800 bases pairs. More extensive exonuclease treatment resulted in a reduction in the frequency of circularization. To determine whether the terminally repetitive regions themselves contained self-annealing sequences that were precluding circularization of more extensively digested DNA, the terminal fragments from HinIII restriction endonuclease digests were isolated, denatured, and tested for their ability to self-anneal. The results of hydroxyapatite column chromatography and electron microscope examination of the terminal regions are consistent with this hypothesis.