During herpes simplex virus type 1 (HSV-1) latent infection in human dorsal root ganglia, limited viral transcription, which has been linked to HSV-1 reactivation ability, takes place. To study the involvement of this transcription in HSV-1 replication in neuronal cells and consequently in viral latency, we constructed stably transfected neuronal cell lines containing (i) the entire HSV-1 latency transcriptionally active DNA fragment, (ii) the same DNA sequence with deletions of the latency-associated transcript (LAT) promoters, or (iii) the DNA coding sequence of the LAT domain. Replication of HSV-1 or a LAT-negative mutant was markedly repressed in the LAT-expressing cells, a phenomenon mediated by the LATs. To study the mechanism responsible for this effect, we examined LAT influence upon expression of HSV-1 immediate-early (IE) genes ICP0, ICP4, and ICP27, by Northern blot analysis. Following infection of a LAT-expressing neuronal cell line with a LAT-negative mutant, the steady-state levels of all three IE mRNAs were reduced compared to those for control cells. Transient transfections into a neuronal cell line indicated that the LAT suppressive effect upon ICP0 mRNA was mediated directly and was not due to the LAT effect upon the ICP0 promoter. We therefore propose that the LATs may repress viral replication in neuronal cells by reducing IE gene mRNA levels and thus facilitate the establishment of HSV-1 latency in nervous tissue.