Vpu enhances HIV-1 release by suppressing CD317-mediated tethering of virions to the cell surface. In HeLa H1 cells, Vpu(+) infection produces significant amounts of tethered virions, although efficient virus release requires Vpu. We have analyzed membrane targeting and assembly of newly synthesized Gag in infected HeLa H1 cells by quantitative pulse-chase assays in both Vpu(+) and ΔVpu virus backgrounds and in the presence and absence of CD317. Our results show that formation of tethered virions precedes release of viruses to the culture medium in the Vpu(+) infection, and CD317 knockdown reduces tethering in both Vpu(+) and ΔVpu virus backgrounds. Significantly, our results indicate that tethered Vpu(+) viruses represent precursors for extracellular viruses, and Vpu thus appears to reverse tethering in HeLa H1 cells after budding.