Restriction endonuclease analysis of viral DNA extracted from wild-type and temperature-sensitive mutants of vaccinia IHD-W (Dales et al., 1978) revealed sequence alterations in approximately 20% of all ts clones examined. The rearrangements were due to deletions up to 250 nucleotide pairs long. Using Eco RI, Sal I, Bam I, Hpa I and Ava I, the deletions were always observed in the same fragments, while analysis with Hind III demonstrated deletions of identical size in the two terminal fragments. Since vaccinia virus contains inverted terminal repeats of more than 10 kb, these clones possess identical deletions of opposite orientation at both ends of the genome. Analysis of several revertants of the ts mutants demonstrated that the deletions probably arise as events independent from those producing ts lesions and are generated spontaneously at high frequency. This implies that a single event during replication caused the elimination of nonessential information, and suggests that circular intermediates must exist transiently during viral replication.