Simian immunodeficiency viruses (SIVs) have an envelope (Env) glycoprotein with an unusually long cytoplasmic domain of 164 amino acids. In this article, we have characterized a series of SIV Env truncation mutants in which the cytoplasmic domain was progressively shortened from its carboxyl terminus by 20 amino acids. Expression by means of the vaccinia virus system showed that all of the SIV Env mutants were expressed and processed into the surface and transmembrane (TM) subunits. When the ability of the Env mutants to associate with SIV Gag particles was examined, we found that deletion of 20 to 80 residues from the carboxyl terminus of the SIV TM cytoplasmic tail abrogated the incorporation of the Env glycoprotein into particles. By contrast, further truncation of the SIV TM protein by 100 to 140 amino acids restored the ability of the Env protein to associate with Gag particles. Interestingly, mutants bearing a 44- or 24-amino acid cytoplasmic domain were incorporated at levels significantly higher than those of the wild-type Env. Single-cycle infectivity assays showed that Env mutants bearing cytoplasmic tails of 144 to 64 amino acids were highly inefficient at mediating virus entry. By contrast, truncation of the cytoplasmic domain to 44 or 24 amino acids drastically enhanced virus infectivity with respect to that conferred by the full-length Env protein. Our results demonstrate that small variations in the length of the SIV Env cytoplasmic domain dramatically influence Env-mediated viral functions.