We have developed a unique multiple step procedure to inactivate human immunodeficiency virus chemically with a very high safety margin while retaining antigenically active structural virion proteins, including gp120, in the final immunogen. The whole virus preparation (1-10 micrograms per dose) was highly immunogenic in a variety of small mammals and induced antibodies that recognized homologous and heterologous strains of HIV-1. Sera from immunized animals bound to peptides representing the entire sequence of the external glycoprotein gp120. Neutralizing antibodies active against the homologous immunizing strain and against heterologous HIV-1 strains were also elicited. Sera with virus neutralizing activity did not bind to MHC class I proteins derived from the human cell line used to grow the virus.