Through mutagenesis study, we have mapped the determinants of sensitivity to Vpu to several sites within the transmembrane domain of agmBST-2, with deletion of the 22-LL-23 residues conferring the highest degree of resistance to Vpu. This resistance activity is further shown as a result of a weak association of agmBST-2 or the hΔLL22/23 mutant with Vpu and a refraction of these BST-2 proteins to down-modulation by Vpu. With the aim of assessing the involvement of the proteasome and the lysosome degradation pathways in Vpu-mediated down-modulation of hBST-2, specific proteasome inhibitors and lysosome inhibitors were tested for their ability of rescuing hBST-2 expression in the presence of Vpu. The results show that the proteasome inhibitors ALLN and MG132 restore hBST-2 expression to the control level, whereas, lysosome inhibitors chloroquine (CQ) or bafilomycin A1 (BafA1) partially blocks down-modulation of hBST2 by Vpu. To determine whether ubiquitination serves as the signal that triggers hBST-2 down-modulation, we first co-transfected the HA-ubiquitin DNA together with hBST-2 DNA in the presence or absence of Vpu. A specific HA-ubiquitin signal was not observed for hBST-2 with Vpu expression. Moreover, when the only two lysine residues (K18 and K21) within the cytoplasmic domain of hBST-2 were mutated, the K(18,21)R mutant is still sensitive to Vpu-induced down-modulation and is unable to restrict production of wild type HIV-1.