Twenty-three strains of herpes simplex virus type 1 were compared for their pathogenicity in 4-week-old BALB/c mice after peripheral (footpad) or intracerebral inoculation. Among those strains examined were (i) six clinical isolates of brain or cerebrospinal fluid origin, (ii) seven clinical isolates of oral or genital origin, (iii) five prototype laboratory strains that have been passaged numerous times in culture, and (iv) five syncytial variants capable of producing cell fusion in culture. Based on comparative 50% lethal dose values, the strains appeared to segregate into one of three classes of neurovirulence. Class I strains were highly virulent by both the peripheral and intracerebral routes of inoculation, class II strains were highly virulent by the intracerebral route only, and class III strains were highly attenuated by both routes of inoculation. In vivo growth curves for whole brain homogenates infected with class III strains revealed titers of infectious virus approaching those found in the brains of animals infected with class I or II strains. These results would therefore suggest that (i) a strain-dependent variation in neural spread exists that may influence the ability of the virus to cause acute neurological disease and (ii) the amount of infectious virus present within an infected brain does not necessarily determine or reflect the clinical status of the animal. Of the clinical isolates examined, the strains recovered from brain tissue of humans after fatal episodes of encephalitis were found to be no more neurovirulent in mice than the strains isolated from nonneural sites. However, although syncytial variants were found to be highly attenuated by the peripheral route, as a group these strains proved to be among the most virulent when inoculated directly into the central nervous system.