The virulence of different vaccine and wild-type yellow fever (YF) viruses for young adult mice was compared using both intracerebral and intranasal routes of administration. Thirty-five different YF viruses killed mice within 12 days following intracerebral inoculation. In contrast, only seven of those examined killed mice following intranasal administration; these were Asibi virus (YF-AS), the French neurotropic vaccine, two out of three 17DD vaccine substrain viruses (Brazil, Colombia but not Dakar) and three out of six wild-type isolates (YF-B7, YF-B12 and YF-B15). None of eight distinct preparations from the 17D-204 vaccine substrain was virulent by the intranasal route. Thus, strains of YF virus can be distinguished on the basis of their virulence for mice if the intranasal route is used. Evidence of heterogeneity in mouse virulence within the populations was obtained using large and small plaques selected from wild-type stocks of virus. Following intranasal inoculation, a YF-AS small plaque variant was more virulent than either its parent virus or a large plaque variant. On the other hand, a large and a small plaque variant from a nonvirulent wild-type strain could not be distinguished in these tests.