Although most viral vaccines used in humans have been composed of live attenuated viruses or whole killed viral particles, the latter approach has received little attention in research on experimental primate immunodeficiency virus vaccines. Inactivation procedures involving heat or formalin appear to adversely affect the viral envelope proteins. Recently we have inactivated human immunodeficiency virus type 1 (HIV-1) with the compound 2,2'-dithiodipyridine (Aldrithiol-2, Aldrich, Milwaukee, WI), which inactivates infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs. HIV-1 inactivated with Aldrithiol-2 retained the conformational and functional integrity of the viral and virion-associated cellular proteins on the viral membrane. We have extended our studies of zinc finger targeted inactivation to simian immunodeficiency virus (SIV) and evaluated the feasibility of applying the procedures to large scale (>30 l) production and purification of the primate immunodeficiency viruses. There was no detectable residual infectivity of SIV after treatment with 1 mM Aldrithiol-2 (>5 logs inactivation). Treatment with Aldrithiol-2 resulted in extensive reaction with the nucleocapsid protein of treated virus, as shown by immunoblot and high-performance liquid chromatography (HPLC) analysis. As expected, the virion gp120SU appeared to be completely unreactive with Aldrithiol-2. Sucrose gradient purification and concentration procedures resulted in little loss of viral infectivity or virion-associated gp120SU. When tested in a gp120-CD4 dependent cell binding assay, the inactivated virus bound to cells comparably to the untreated virus. Analysis of gp120-CD4 mediated postbinding fusion events showed that the inactivated virus could induce CD4-dependent fusion with efficiencies similar to the untreated virus. Inactivation and processing of primate immunodeficiency viruses by methods described here results in highly concentrated virus preparations that retain their envelope proteins in a native configuration. These inactivated virus preparations should be useful in whole killed-particle vaccine experiments as well as laboratory reagents to prepare antisera, including monoclonal antibodies, and to study noninfective virion-cell interactions.