The role of the cytoplasmic tail of the Moloney murine leukemia virus transmembrane protein in the regulation of syncytia was examined. Three mutations within the cytoplasmic tail were studied. Linker-insertionin7705-12a is within the viral-associated cytoplasmic tail, linker-insertionin7748-12a is within the R peptide, and a third mutation expresses TM lacking the R peptide (Env R − ). The Env R − construct was nonviable in Rat1 cells, however, rapidly reverted to a form containing the R peptide when passaged in NIH/3T3 cells.in7705-12a was temperature-sensitive in Rat1 cells, as previously characterized, but was viable at either temperature in NIH/3T3 cells.in7748-12a was comparable with wild-type M-MuLV. The ability of theenvconstructs to form large multinucleated syncytia with NIH/3T3 and XC cells were examined using transient expression assays, eliminating reversion events due to viral passage and reverse transcription. The Env R − constructs formed syncytia with NIH/3T3 cells.in7705-12a displays enhanced proteolytic cleavage of the R peptide. Neither linker-insertion mutationin7705-12a orin7748-12a activated fusion with NIH/3T3, despite the abundance of processed TM within7705-12a. All three mutants were fusion competent with Rat XC cells, even in the absence of any cleavage of the R peptide. These results provide insights regarding steric and the temporal affects of cleavage of the R peptide and the assembly of a fusion competent oligomer.