Synchronous reactivation of bovine herpesvirus type 1 in all latently infected rabbits was achieved following a single intravenous dose of dexamethasone. Reactivated latent virus was first present in ocular secretions between 48 and 72 h post-dexamethasone treatment (PT). Cell-free infectious virus, viral-antigen-containing neurons, and pathologic changes were detectable in trigeminal ganglia (TG) by 48 h PT. A shift from the viral transcriptional pattern characteristic of the latent state (latency-related RNA [LR RNA]) to one typical of that seen during acute infection was detected in a small number of neurons in latently infected TG between 15 and 18 h PT, with viral DNA first detectable by in situ hybridization at 18 to 21 h PT. The number of LR RNA-containing neurons in latently infected TG decreased significantly at 24 and 48 h PT but returned to near-normal levels by 72 h PT. Correlation of this decrease with viral reactivation suggests that altered regulation of LR RNA transcription is a significant event in the process of viral reactivation.