Purified and concentrated Venezuelan equine encephalitis (VEE) virus derived from tissue cultures, rendered noninfectious by ionizing radiation with retention of in vitro serological activity, also retained a high level of immunogenicity. In mice, fluid vaccines afforded excellent protection against lethal challenge with homologous Trinidad strain VEE virus. A direct relationship was observed between concentration of vaccine or number of injections and survival. One intraperitoneal inoculation of undiluted vaccine protected essentially all mice challenged 21 days later with 100,000 mouse intraperitoneal LD50 of virus. Similarly, mice receiving three injections of vaccines diluted 1:100 were completely protected. Noninfectious VEE virus preparations combined with adjuvant 65, a nontoxic metabolizable vehicle, were likewise very effective in protecting mice immunized intraperitoneally or subcutaneously against lethal challenge. Guinea pigs immunized subcutaneously with adjuvantcombined vaccine survived lethal challenge of 1,000,000 guinea pig intraperitoneal LD50.