Vaccinia virus (VV) is one of the largest and most complex of animal viruses, with a virion that contains about 100 different polypeptides. Assembly of the viral proteins occurs in discrete cytoplasmic sites leading to formation of two infectious forms, an abundant (>90%) intracellular mature virus (IMV) with an envelope, and a minor extracellular enveloped virus (EEV) with an extra membrane acquired from the trans-Golgi network. It has been shown that while EEV contains in the outer membrane cellular proteins probably acquired during virus release from cells, however, IMV exclude host proteins during assembly. Since VV recombinants (VVr) expressing genes of interest are candidates as potential vaccines against pathogens and cancer, it becomes important to know if VVr can acquire foreign proteins during morphogenesis. In this investigation we show that purified virions (IMVs) from VVr can incorporate foreign proteins into different sites in the virus particle. By sequential fractionation of virion compartments with detergents, we found foreign proteins in the lipid envelope (cytokine IL-12 and CS antigen of Plasmodium yoelii), as part of a protein matrix-like membrane (HIV-1 gp41 of env), or more closely associated with the core containing the DNA complexes (HIV-1 gp160; a multiepitopic protein with the V3 loop of HIV-1 Env from different isolates, and beta-galactosidase). Similar findings were observed with purified virions derived from the WR strain as well as from the highly attenuated modified vaccinia virus Ankara (MVA) strain. These observations should be taken into consideration when VVr are used in clinical trials or in other vaccination approaches.