Activation of the murine leukaemia virus (MLV) envelope protein (Env) requires proteolytic cleavage of the R-peptide, a 16 amino acid C-terminal part of the cytoplasmic tail (C-tail) of Env. This paper demonstrates the presence of R-peptides in Env proteins of C-type retroviruses of simian, avian and porcine origin. Sequence alignment with the MLV C-tail led to the identification of a conserved hydrophobic protease cleavage motif located in the centre of retroviral Env protein C-tails. Expression of Env proteins, truncated at the predicted cleavage sites, of spleen necrosis virus (SNV), gibbon ape leukaemia virus and porcine endogenous retroviruses resulted in cell-cell fusion as monitored by microscopy and reporter gene fusion assays. Western blot analysis of MLV particles pseudotyped with the SNV Env protein demonstrated proteolytic cleavage of the SNV R-peptide by the MLV protease. Our data suggest that activation of membrane fusion by R-peptide cleavage is a common mode in C-type retroviruses.