The herpes simplex virus (HSV) infected cell protein 8 (ICP8) is required for viral DNA replication and normal viral gene expression. Previous work in our laboratory has shown that ICP8 may play a role in stimulating late gene expression. In V2.6 cells which express the d105 mutant form of ICP8, synthesis of late proteins and accumulation of the late gC mRNA are reduced during HSV infection (Gao, M., and Knipe, D.M., J Virol. 65, 2666-2675, 1991). To determine if the negative effect of d105 ICP8 on the late gene expression was exerted at the transcriptional level, we measured the levels of mRNAs and transcription from three late genes, gC, UL47, and gD, in V2.6 cells and Vero cells infected with the HSV-1 wild-type virus. In infected V2.6 cells, the levels of late gC and UL47 mRNA were 7- to 12-fold lower than those of infected Vero cells under conditions where the levels of viral DNA replication in these two cell types were similar. The transcription levels of these two late genes in infected V2.6 cells were reduced to similar extents (9- to 14-fold). The levels of accumulated mRNA and transcription of the early-late gD gene also showed parallel reductions in infected V2.6 cells (about 6-fold). In contrast, transcription of the beta pol gene was reduced only slightly (about 2-fold) by d105 ICP8. These results demonstrate that the d105 ICP8 inhibits expression of three viral late genes at the transcriptional level, and in general, the effect of d105 ICP8 on viral gene expression appears to correlate with the extent to which expression of the gene is stimulated by viral DNA synthesis.