Both rate zonal centrifugation and gel electrophoresis have revealed that the mature double-stranded DNA of bacteriophage T5 has single-stranded breaks (nicks) at specific sites. Neither of these procedures has previously revealed site-specific nicks in the double-stranded DNA of other bacteriophages, including T7. In the present study, denaturing gel electrophoresis, followed by specific DNA detection, reveals that a small fraction of mature T7 DNA molecules, like most T5 DNA molecules, has site-specific nicks. The procedure of specific detection is to probe with an oligonucleotide specific for one of the ends of T7 DNA. If position 0.0 is the left genetic end and position 100.0 is the right genetic end of T7 DNA, the nicks on the 5' left-oriented strand are at 11.3, 12.4, 65.7, 79.2, and 86.0; the nicks on the 5' right-oriented strand are at 23.3 and 26.5 (+/- 0.5). The positions of the three rightmost nicks are indistinguishable from those of double-stranded breaks that produce previously demonstrated shorter than mature length DNAs packaged in vivo. We propose that the T7 nicks are produced by premature activity of the T7 terminase during DNA packaging.