Integrated retroviral DNA is flanked by short direct repeats of the target DNA. The length of these repeats is specific for the provirus that is integrated (H.E. Varmus, in J.A. Shapiro, ed., Mobile Genetic Elements, 1983). For the human immunodeficiency virus type I (HIV-1), the length of the direct repeats in the target DNA was shown to be 5 bp in one case (Muesing et al., Nature [London] 313:450-458, 1985) and 7 bp in another (Starcich et al., Science 227:538-540, 1985). One possible explanation for this discrepancy is that the direct repeats flanking HIV-1 proviruses are variable. To investigate this, we analyzed the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. In each clone the provirus was flanked by a 5-bp direct repeat of human DNA. Analysis of the proviral clone previously described as being flanked by a 7-bp direct repeat of target DNA (Starcich et al., op. cit.) revealed that this clone was flanked by a 5-bp repeat instead. Therefore, we conclude that HIV-1 proviruses are flanked by 5-bp direct repeats of human DNA. The sequences of the 5-bp duplications from the different proviral clones do not have any apparent similarity to each other or to HIV-1 DNA.