C-57 albino weanling mice were latently infected with herpes simplex virus (Mp strain, type 1) by inoculation of 10(4) plaque forming units in the right hind footpad. The virus was demonstrable in explant cultures of the sacral dorsal root ganglia of these mice for as long as 18 months following inoculation. In addition, the virus was detectable when homogenates of these latently infected ganglia were placed on to differentiated organotypic cultures of fetal mouse dorsal root ganglia for as long as 8 months following inoculation of the mice. Virus was not demonstrable in these homogenates when they were placed on the Hela cells. The results suggest that during herpes simplex virus latent infection in mice there is continuous synthesis of infectious virus, probably in a highly localized area, which is detectable if a sensitive indicator substrate, such as these organotypic cultures, is used.