Herpes simplex virus type 1 (HSV-1) is transcriptionally active during latent infection in human peripheral sensory ganglia. This transcription has been linked to the ability of the virus to reactivate, but its potential gene products and mechanisms of action are unknown. To analyze the viral latency-related transcripts in neuronal and non-neuronal cell lines in an isolated cellular system, a 10.4 kb DNA fragment, which covers the entire viral transcriptionally active latency-associated region, was cloned under control of the constitutive cytomegalovirus promoter (pNM3). During transient transfection of a human embryonic kidney 293 cell line, pNM3 expressed high levels of the 2.0 kb latency-associated transcript (LAT) that was not polyadenylated. The 1.5 kb LAT as well as the minor hybridizing RNAs could not be identified by Northern blotting analysis. pNM3 expression was further analyzed following transfection of two neuronal, C1300 and ND7 cell lines. The 2.0 kb LAT was synthesized at high levels in these cell lines. The 1.5 kb LAT, which in vivo can be identified only during HSV-1 latent infection in tissues which facilitate reactivation, was present at very low amounts in 293 and C1300 cells using reverse transcription PCR analysis. Higher amounts of the 1.5 kb LAT were produced in ND7 cells, a neuronal cell line shown to possess neuronal-specific splicing proteins. However, the 1.5 kb LAT was present in ND7 cells in lesser amounts than produced during latent infection in peripheral sensory ganglia. This novel cellular system provides now a tool for future studies of the role of the 1.5 kb and the 2.0 kb LATs in HSV-1 latency.