Two preparations of yellow fever vaccine (17D) were studied by clonal analysis. From one (17D-England) three types of clones were isolated, each differentiated by plaque size in Vero cells and virulence for intracerebrally inoculated mice: small plaque (SP) and large plaque (LP) clones were lethal, whereas medium plaque (MP) clones failed to kill at doses up to 10(6) p.f.u. Analysis of 24 randomly selected clones showed the original vaccine to be a mixture of predominantly MP and SP variants; 58% and 42% respectively. A single passage in suckling mice modified this composition to 90% SP and 10% LP variants. The response of six inbred strains of mice to intracerebral inoculation of the MP variant varied from complete resistance to complete susceptibility. From another 17D substrain (17D-South Africa) two types of variants were isolated (a SP avirulent type and a LP type of intermediate virulence), both different from the previously described variant.