A molecular hybridization technique using biotinylated DNA probes was used to detect bovine herpesvirus 1 (BHV-1) nucleic acid species immobilized on nitrocellulose. Seventeen recombinant plasmids containing HindIII restriction fragments of the BHV-1 genome were compared for their ability to detect immobilized BHV-1 DNA from purified virus and infected cells. One probe, pCB2, labeled by nick translation with either 3H or biotin, detected as little as 10 pg of viral DNA. In time course experiments, BHV-1 DNA could be detected by 2 h postinfection in 10(6) infected cells. BHV-1 DNA was detected in nasal swabs and exudate from experimentally infected cattle, even when specimens had been stored for over a year. In a retrospective study of a respiratory disease outbreak in a feedlot, hybridization was compared with virus isolation for diagnosis of BHV-1 infections. The sensitivity rate was 0.68 with virus isolation as the referent standard. Blot hybridization provides a novel approach with unique applications for the diagnosis of bovine herpesvirus infections.