The vaccinia virus forms two morphologically distinct infectious virus particles: intracellular mature virus (IMV) and extracellular enveloped virus (EEV). The envelope of EEV is a Golgi-derived membrane (wrapping membrane). A mutant (vRB10) lacks the ability to form the EEV. In medium containing a neutralizing antibody (2D5mAb), the vRB10 mutant was diluted out from infected cells, whereas the IHD-J strain of vaccinia virus replicated well. The result indicated that the 2D5mAb specifically neutralized the IMV. The 2D5-resistant EEV appeared at 6-7 hr postinfection, and over 65% of infectious virus in the culture fluid was EEV at 48 hr after infection. The EEV was resistant not only to the 2D5mAb but also against several neutralizing antibodies, including polyclonal antivaccinia serum reactive with proteins of the wrapping membrane. Freeze-thawing and other procedures that may damage the wrapping membrane converted the EEV to a form susceptible to the antibodies. Since specific infectivity was not affected by the damage or by exposure to antibody against the wrapping membrane proteins, the wrapping membrane did not directly participate in penetration. The infection process of vaccinia virus was analyzed by comparison of responses to acid treatment between normal IMV and trypsin-treated IMV. Proteolytically activated IMV infected rapidly responding to acid. The protected form virus, which was noninfectious under usual conditions, was proteolytically activated on cell membrane then responded to the acid. Proteolysis activated the virus, and an acidic condition accelerated fusion between the activated IMV and plasma membrane. The virus in the EEV wrapping membrane was the activated form that has the capacity to fuse with the cell membrane. However, the infection of intact EEV was more sensitive against lysosomotropic agents (NH4Cl, neutral red) than that of the trypsin-activated IMV. Resistance to the 2D5mAb, sensitivity to lysosomotropic agents, and acceleration of infection by acid suggested that the intact EEV penetrated by virus-endosome membrane fusion. The combined effect of the presence of wrapping membrane and the process of internalization via an endocytic mechanism rendered EEV resistant to neutralizing antibodies.