Pseudorabies virus (PRV) is a porcine herpesvirus that establishes latent infections in trigeminal ganglia. To determine whether PRV expresses any transcripts that could play a role in latency, the trigeminal ganglia of 14 pigs previously inoculated through the nose and latently infected with PRV(Ka) were assayed by in situ nucleic acid hybridization for the presence of PRV-specific RNA. Hybridizations employing probes encompassing the entire viral genome revealed that an area extending from 0.64 to 0.82 map units was transcriptionally active. The DNA probe that most consistently detected transcripts was BamHI-8, a fragment which contains the gene for the immediate-early protein. With this probe, ganglia from 10 (71%) of 14 pigs scored positive for PRV RNA, although only 1 (8%) of 12 of the ganglia from the opposite side reactivated virus after explanation and culture of latently infected trigeminal ganglia. The RNA was transcribed from the strand opposite to that coding for the immediate-early protein; the signal was neuronally localized, with dense nuclear accumulation accompanied by variable numbers of grains over the cytoplasm. Northern RNA blot analysis showed that a discrete set of poly(A)- PRV transcripts were present in latently infected trigeminal ganglia. Additional in situ nucleic acid hybridization analysis revealed that the 3' limit of the transcriptionally active area was located in a 1.2-kilobase fragment upstream and adjacent to the 5' end of the immediate-early protein RNA, whereas the 5' limit was as much as 4.9 kilobases downstream from the 3' end of this RNA. PRV therefore expresses latent-phase transcripts that, although similar in many respects to latent-phase transcripts reported for other herpesviruses, have some unique properties.