Extracts of vaccinia-infected HeLa cells were rendered free from infectious virus by centrifugation followed by membrane filtration and were shown to be toxic to uninfected HeLa cells in the presence of hypertonic MgSO4, used as a macromolecular uptake inducer, under conditions which did not kill control cells. Extracts from uninfected cells were nontoxic. This biological test was adapted to a semi-quantitative assay which was used to monitor the purification of the cytotoxic factor by DEAE-cellulose and Sephadex G-100 chromatography. The cytotoxic factor was purified 100-fold, shown to be of molecular weight 30 -- 100,000 daltons, acidic and completely inactivated by soluble trypsin but not by ribonuclease under conditions believed to degrade both single- and double-stranded RNA species. It was demonstrated to be virus specific by approrpiate immunosorbent chromatography. Extracts were also prepared from vaccinia-infected HEp-2, RK and W-K cells respectively. A virus-specific factor, toxic to uninfected HeLa cells, with similar chromatographic properties to that isolated from infected HeLa cells, was isolated from these three additional cell lines. The concept of virus induced cytotoxins, substances which exert their toxic effect in the host cells in which they are made, is discussed.