Our studies aim to elucidate the functions carried out by the very long, and in its length highly conserved, C-terminal cytoplasmic domain (Env-CT) of the HIV-1 glycoprotein. Mass spectrometric analysis of cellular proteins bound to a tagged version of the HIV Env-CT led to the identification of the prohibitin 1 and 2 proteins (Phb1 and Phb2). These ubiquitously expressed proteins, which exist as stable heterodimers, have been shown to have multiple functions within cells and to localize to multiple cellular and extracellular compartments. The specificity of binding of the Phb1/Phb2 complex to the Env-CT was confirmed in various manners, including coimmunoprecipitation with authentic provirally encoded, full-length Env. Strong binding was dependent on Env residues 790 to 800 and could be severely inhibited by the double mutation L799R/L800Q but not by mutation of these amino acids individually. Analysis of the respective mutant virions revealed that their different abilities to bind Phb1/Phb2 correlated with their replicative properties. Thus, mutated virions with single mutations [HIV-Env-(L799R) and HIV-Env-(L800Q)] replicated similarly to wild-type HIV, but HIV-Env-(L799R/L800Q) virions, which cannot bind Phb1/Phb2, exhibited a cell-dependent replicative phenotype similar to that of HIV-Env-Tr712, lacking the entire Env-CT domain. Thus, replicative spread was achieved, although somewhat delayed, in "permissive" MT-4 cells but failed to occur in "nonpermissive" H9 T cells. These results point to binding of the Phb1/Phb2 complex to the Env-CT as being of importance for replicative spread in nonpermissive cells, possibly by modulating critical Phb-dependent cellular process(es).