L1 element DNA increased over time in both total and high-molecular weight DNA from HIV-1-infected cells, consistently reaching 2-3 fold above baseline within 144 hours. By absolute quantitation this was determined to represent the generation of tens of thousands of novel L1 sequences per cell. By sorting Gag+ from Gag- cells we confirmed that this increase was primarily restricted to HIV-1 infected cells. Using TGDA we identified de novo LINE-1 insertions in HIV-1 infected primary CD4+ T cells. These sequences displayed the hallmarks of L1 retrotransposition events including long poly(A) tracts at the insertional junction, and were absent in DNA from autologous uninfected cells. The eGFP reporter retrotransposition assay showed an increased L1 retrotransposition frequency in HIV-1 infected Jurkat cells as compared to uninfected controls. Infection with HIV-1 ΔVif resulted in L1 retrotransposition frequences which were substantially less than that observed with wild-type virus, but still elevated as compared to uninfected controls (36.0 +/- 1.2% eGFP+ for wild-type infection, versus 18.4 +/- 0.5% for HIV-1 ΔVif, and 6.3 +/- 0.7%, p < 0.0001 for either comparison).