Biotin-labeled DNA probes for bovine herpesvirus type 1 (BHV-1) were used to detect viral nucleic acids in infected cell cultures and clinical specimens by in situ hybridization. Hybridization signal was detected 2 hours after inoculation in the cytoplasm of infected cells, presumably representing input virus. Hybridization was first detected in the nucleus at 4 hours after inoculation; by 10 hours after inoculation, hybridization signal was detected in both nucleus and cytoplasm of almost 50% of the cells. By 15 hours after inoculation, 95% of the cells were positive. Treatment of specimens with ribonuclease or deoxyribonuclease before hybridization allowed clear differentiation of virus-specified DNA and RNA within infected cells. The BHV-1 nucleic acid sequences were detected in nasal epithelial cells obtained from inoculated calves. Since in situ hybridization provides a rapid technique for the detection of BHV-1-specified nucleic acid sequences, it should facilitate studies on the replication, pathogenesis, and diagnosis of BHV-1 infections.