The temperature-sensitive BHK21 hamster cell line tsBN67 ceases to proliferate at the nonpermissive temperature after a lag of one to a few cell divisions, and the arrested cells display a gene expression pattern similar to that of serum-starved cells. The temperature-sensitive phenotype is reversible and results from a single missense mutation--proline to serine at position 134--in HCF, a cellular protein that, together with the viral protein VP16, activates transcription of herpes simplex virus (HSV) immediate-early genes. The tsBN67 HCF mutation also prevents VP16 activation of transcription at the nonpermissive temperature. The finding that the same point mutation in HCF disrupts both VP16 function and the cell cycle suggests that HCF plays a role in cell-cycle progression in addition to VP16-dependent transcription.