Tracheal organ culture (TOC) techniques utilising multiple-well plastic trays were used to detect and assay latent infection established by infectious laryngotracheitis (ILT) herpesvirus in clinically normal chickens. Between 3 and 16 months after tracheal exposure to wild strain (CSW-1, haemorrhagic tracheitis) ILT virus and 2 to 10 months after exposure to vaccine strain ILT (SA-2), groups of chickens were examined for evidence of infection. Neither the examination of tracheal swabbings in monolayer cell cultures nor the inoculation of tracheal tissue suspensions detected virus, and this result was not influenced by preliminary immunosuppressive treatment of the birds with cyclophosphamide or dexamethasone. Latent infection was detected, however, by TOC in 6 (38%) of 16 chickens 100 days to 15 months after exposure to wild strain ILT virus and in 4 (44%) of 9 chickens 2 to 10 months after exposure to the vaccine strain. These data provide the first proof that both wild and vaccine strains of ILT virus regularly establish long term latent infections. Sites of establishment of latent infection in the trachea were highly focal in distribution. Virus reactivation was demonstrated in only 20 (8.3%) of the TOC preparations established from previously infected chickens and usually from only one or two sites in each trachea. Both strains of ILT virus exhibited characteristics of latency in vitro in that virus was not detectable in supernatant fluids until 5 to 6 days after establishment of TOC. Virus shedding then usually continued for 1 to 2 weeks 10(2) to 10(4) PFU/ml being produced each 2 to 3 days. In some preparations, virus production continued for up to 30 days.