In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse–chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGPer, full-length GP with oligomannosidic N-glycans, was detected. preGPer (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP1,2, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP1, and the carboxyl-terminal 26-kDa fragment GP2. GP1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.