The newly discovered vacuolating virus, S.V.40, appears to be a common and essentially ubiquitous contaminant of rhesus monkey kidney cell cultures and a likely common contaminant of cynomolgus kidney cultures in which it develops to high titer without evident cytopathic change. The virus grows readily in grivet, vervet and patas kidney and in rhesus testicle cultures causing a unique cytopathology characterized by ballooning and intense vacuolation. Some biological and biophysical properties of the virus are described. Principal interest in and importance of the vacuolating virus arise from its demonstrated presence in all 3 types of Sabin''s live poliovirus vaccines and in numerous respiratory virus seed lines employed in human volunteer inoculation experiments. Demonstration of the virus has raised the question of safety following administration to human subjects, especially infants, and of possible lack of validity of conclusions reached from experiments to measure neurovirulence or "genetic" markers of attenuated poliovirus vaccine strains and to determine the pathogenicity of respiratory viruses for man. The vacuolating virus is effectively dealt with in killed virus vaccines by virtue of its susceptibility to formalin. The optimal solution to the live virus vaccine problem appears to lie in elimination of the virus from such preparations.