The Vpu protein of HIV-1 induces degradation of CD4 in the endoplasmic reticulum. Previous studies have elucidated the role of the CD4 cytoplasmic domain in the Vpu-induced degradation process, and the minimal Vpu responsive element mapped to a small region in the CD4 tail. In the present study, we have carried out both biochemical and biological experiments to analyze the role of the CD4 anchor domain in the Vpu-induced degradation process. We generated chimeric proteins that possessed the ecto-anchor domains of gp160 and the cytoplasmic domain of CD4. The chimeric envelope glycoproteins were functionally active in the fusion of HeLa CD4+ cells, with the exception of those having the arginine to isoleucine (R to I) substitution in the gp160 anchor domain. Coexpression studies revealed that these chimeric glycoproteins were stable and functionally active in the presence of Vpu, as opposed to those having the anchor-cytoplasmic domains of CD4. The half-life of Vpu-sensitive chimeric glycoproteins was calculated to be approximately 60-90 min, whereas Vpu-resistant envelope glycoproteins exhibited relatively longer half-lives in the presence of Vpu. Taken together, these studies strongly suggest that the CD4 anchor domain appears to provide critical sequence or structural elements through which the Vpu protein could access CD4 or glycoproteins bearing the Vpu responsive element for degradation in the endoplasmic reticulum.