Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (1) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (2) the cleavage of viral DNA concatemers was coupled to packaging, (3) the a sequence contained the information required for its own amplification, and (4) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.