To obtain further information on the latent infection with bovine herpesvirus 1 (BHV-1) in the natural host, we examined the nasal secretions and various tissues of experimentally infected calves using virus isolation, polymerase chain reaction (PCR), and immuno-PCR/antigen (Immuno-PCR/Ag) assays. In the nasal secretions, viral DNA was detected in samples with virus isolation titers of 10(4.3) TCID50 or more by PCR. On the same samples, Immuno-PCR/Ag remained positive up to day 19 p.i. the last day of test. BHV-1 DNA was detected from the following tissues in all the calves at day 22 p.i.: trigeminal ganglia, ovaries, lungs, nasal and tracheal mucosae, spleen, prescapular and precrural lymph nodes, and peripheral blood leukocytes (PBL), but not skeletal muscles. Virus was not recovered from any of these tissues. The present findings suggest a possible role of leukocytes in BHV-1 latent infection.