Focus formation by Rous sarcoma virus (RSV) was significantly enhanced when virus was incubated with the saline fraction of chloroform extracted chicken egg yolk, prior to infecting chicken embryo cells. The enhancement was restricted to members of RSV subgroups B, C and D and was proportional to yolk dilution. Subgroup A virus was never affected. In all, 108 yolk samples from specific pathogen free chickens were investigated. Of these 78 to 97% stimulated focus formation. RSV(RAV-50) of subgroup D which was stimulated up to tenfold, was the most strongly affected strain. The enhancing principle was shown to be a specific yolk factor, and its effect remained constant even after several years' storage. Crude yolk specimens showed essentially the same enhancing property. The chemical nature of the yolk factor is still unknown. It must, however, be taken into account when assaying for avian leukosis virus antibodies.