During reverse transcription the synthesis of plus strand viral DNA is initiated from an RNA polypurine primer immediately upstream of the U 3 region. The polypurine tract (PPT) sequence at this site is in HIV-1 also present in the middle of the genome. Here we demonstrate that a subpopulation of linear unintegrated HIV-1 DNA has a discontinuity in the plus strand within less than 50 bp from this central PPT, consistent with its utilization as a plus strand initiation site.