Vpu is an accessory protein of HIV-1 that is absent in most simian immunodeficiency viruses (SIVs). Vpu enhances HIV particle assembly or release in a variety of human cells, but exerts no effect on assembly in African green monkey (AGM) cells. We carried out experiments to determine whether a Vpu-like “positive” factor exists in AGM cells, or conversely if a restrictive factor is present in human cells. Heterokaryons formed between human and AGM cells revealed the presence of a dominant restriction to assembly that was conferred by the human cells. Addition of Vpu to human-simian heterokaryons relieved the block to assembly. Thus one important result of the acquisition of the vpu gene by a few SIV species and by HIV-1 is the ability to overcome a restriction to assembly in human cells; we speculate that this acquisition may have facilitated cross-species transmission to humans. HIV-2 does not encode vpu, but overcomes the same assembly restriction through encoding an envelope glycoprotein that can functionally complement the particle assembly effects of Vpu. Identification of the subcellular site of action of Vpu should provide important clues to its mechanism of action and may help reveal the nature of the host cell restriction. While Vpu is known to localize to the ER and Golgi, we have found that a subpopulation of cellular Vpu is present in the pericentriolar recycling endosome. Disruption of recycling from this compartment prevented both Vpu and the HIV-2 envelope glycoprotein from enhancing particle release in restrictive human cells. These results indicate that both Vpu and the HIV-2 envelope glycoprotein depend upon the activity of the pericentriolar recycling endosome for their effects on retroviral particle release.