Defective genomes generated during serial propagation of herpes simplex virus type I (Justin) consist of tandem reiterations of sequences that are colinear with a portion of the S component of the standard viral genome. We determined the structure of the novel U(S)-a junction, at which the U(S) sequences of one repeat unit join the a sequences of the adjacent repeat unit. Comparison of the nucleotide sequence at this junction with the nucleotide sequence of the corresponding U(S) region of the standard virus genome indicated that the defective genome repeat unit arose by a single recombinational event between an L-S junction a sequence and the U(S) region. The recombinational process might have been mediated by limited sequence homology. The sequences retained within the U(S)-a junction further define the signal for cleavage and packaging of viral DNA.