Small interfering RNAs have been demonstrated to serve as a molecular defence against numerous retroviruses in plants and insects and, more recently, in primates. With the recent findings of micro-RNAs (miRNAs) that seem to play a pivotal role in the survival of the host, we have explored the role of miRNAs in lentiviral (LV) replication. We have previously hypothesized that, at least in the case of lentivirus infection, small interfering RNAs are involved in the inhibition of these types of viruses by the formation of intramolecular triplex formation (triplexes) between the polypurine tracks sequences of LV provirus and miRNAs and blocking the viral replication at the preintegration complex levels, placing these viruses into a suspended latency. Using several latently and chronically infected LV cell lines and human PBMCs from HIV–1-infected individuals, we show that perinuclear triplexes are formed in LV-infected cells. The number of triplexes decreased in cells with productive replication of LVs. Therefore, the degree of replication of HIV-1 and other LVs, both in the HIV-1 or other LV-infected cell lines and the HIV-1 infected PBMCs, inversely correlate with the number of cytoplasmic triplexes present in a particular cell. This correlation was further confirmed by the stimulation of PBMCs and LV-infected cell lines with appropriate mitogens. Treatment with Tagetin, a RNA polymerase III inhibitor, resulted in a significant decrease in triplexes and a dramatic increase in the LV replication. Our data suggest that triplex formation may be an important mechanism of LV latency mediated by endogenous miRNAs.