Uninfected simian cells (CV-1) produce two different proteins, one of which enhances the infectivity of echovirus 6 and the other enhances the infectivity of SV40 in less susceptible cells. The enhancers are released by metabolizing cells into the culture medium. The SV40 enhancer protein is larger and less acidic than the echovirus enhancer. The SV40 enhancer protein can be dissociated into 2 subunits with apparent molecular weights of 42,000 and 24,000. The echovirus enhancer protein consists of 2 subunits with apparent molecular weights of 28,000 and 31,000. Electrophoretically purified enhancer proteins interact with virions and retain their biological activities and viral specificities. The SV40 enhancer stimulates expression of SV40-T antigen under conditions in which untreated virus does not initiate expression of T antigen. This result and the observation that the enhancers do not increase the infectivity of the nucleic acids of their respective viruses suggest that the enhancers act either at the stage of penetration or uncoating.