Papillomaviruses are believed to play an important role in the development of genital carcinoma. Herpes simplex virus (HSV) has been proposed as a cofactor. Here we show that HSV-1 interferes with the expression of human papillomavirus (HPV-18) genes in HeLa cells by reducing the amount of papillomaviral mRNA. By 7 h after HSV-1 infection, expression was reduced by a factor of 50. Experiments with the HSV-1 mutant tsK, with cycloheximide and with u.v.-irradiated virus indicated that the reduction was not due to newly made immediate early, early or late HSV-1 gene products but rather to a component of the virion. Replication of the HSV-1 is therefore not required for the reduction of the HPV-18 mRNA. The HSV-1 strain 17+, which has only a very weak virion host shutoff function, still specifically decreased the level of the papillomaviral mRNA suggesting that either the decrease is due to a new HSV-1 function or that the HPV-18 mRNA is especially sensitive to the low residual host shutoff activity of strain 17+. Experiments with the virus 17(41-), in which the host shutoff function is inactivated by a mutation in the UL41 gene, showed clearly that it is the host shutoff function which is responsible. The papillomaviral mRNA therefore appears to be hypersensitive to the herpesvirus host shutoff function.