The latency-associated transcripts (LAT), which code from an 8.5 kb segment of the internal repeat region of the HSV genome, are the only viral transcripts that are present during HSV latent infection. However, little is known about the relative contribution of promoter activity, degradative processes, and elements or regions affecting long term expression of these transcripts in latently infected neurons. To begin to address this question we investigated LAT promoter activity during acute and latent infection. Mouse footpads were infected with KOS/62-3, an engineered herpes simplex virus in which both copies of the LAT promoter are used to drive expression of the Escherichia coli lac Z gene. Four days post-inoculation (p.i.) abundant β-galactosidase (β-gal) protein and transcripts were present within ganglionic neurons as assayed by enzyme histochemistry and in situ hybridization. In contrast, by Day 21 (at which time a latent infection had been established) no β-gal transcripts were present in infected ganglia, even when assayed by the polymerase chain reaction (PCR). These findings indicate a significant drop in LAT promoter activity between Day 4 and Day 21 p.i. To provide confirmatory evidence for this conclusion we infected mice with a second viral construct, KOS/67-7, in which the LAT promoter was used to drive expression of the nerve growth factor (NGF) gene. Four days p.i., abundant NGF antigen and transcripts were present in infected ganglionic neurons, but no evidence of transcription of the cloned NGF gene could be found in latently infected ganglia. Our findings suggest that LAT promoter activity is severely restricted during the latent phase of ganglionic infection.