The a sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage) to yield unit-length linear HSV-1 DNA prior to or during packaging of HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and 0.5 kb (longer band), the lengths of which correspond to one and two units of the a sequence, was identified using acrylamide gel electrophoresis of HSV-1 DNA preparations extracted by the method of Hirt. Twelve DNA fragments from each band were molecularly cloned, and nucleotide sequences were determined. Both termini of eight (67%) DNA clones from the shorter band corresponded to the specific cleavage site on DR1. Five (41%) DNA clones from the longer band had a terminus corresponding to the specific cleavage site on DR1 on one side, but not on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA clones from the longer band were in and around DR1. Thus, cleavage events of DR1 can be classified into three categories: (i) authentic cleavage; (ii) site-specific cleavage on the third DR1 distal from the S component (secondary site-specific cleavage), which is related to the generation of the shorter DNA band in combination with authentic cleavage; and (iii) less-specific cleavage events in and around other DR1 elements which relate to the generation of the longer DNA band.