The temporal appearance of the major latency-associated transcript (LAT) of herpes simplex virus, type-1 (HSV-1) was examined in sensory neuronal cultures during the establishment of either a latent or a lytic infection. Under conditions that result in the establishment of a latent infection, a significant delay in LAT accumulation was observed. The delay in the appearance of LAT was reflected in both a reduced number of LAT-positive neurons detected by in situ hybridizations and by low levels of the major 2-kb LAT detected by Northern blot analysis at early times compared to later in the latent infection. The percentage of LAT-positive neurons shown by in situ hybridizations and the relative abundance of the major LAT by Northern blot analysis increased markedly by 14 days after inoculation with virus. In addition to the major LAT, a spliced 1.5-kb LAT species was detected in Northern blot analysis after establishment of latency in the neuronal cultures, similar to observations in vivo. In contrast to the latent infection, under conditions that produced lytic infections in the neuronal cultures, LAT and HSV-1-specific antigens were detected in the majority of neurons 24 hr after inoculation with virus. These results indicate that LAT expression during the establishment of latency is regulated differently than during the lytic infection: LAT expression appears to be inhibited initially during the establishment of latency, whereas LAT is readily expressed during the lytic infection.