We report a simple procedure for the detection of Epstein-Barr virus (EBV) by in situ DNA-RNA hybridization with an alkaline phosphatase-linked oligonucleotide probe. EBV-producing cell lines P3HR-1 and Akata were treated with phorbol ester and n-butyrate, and anti-human IgG, respectively. This treatment resulted in highly increased populations of cells with EBV transcripts of the latent membrane protein 1 (LMP1) and envelop glycoprotein gp350/220, but not of EBV-encoded small nuclear RNAs (EBERs). Synthesis of the LMP1 protein, which was encoded by the induced mRNA, was mostly dependent on viral DNA synthesis, as shown by double or single labeling for in situ DNA-DNA hybridization with the oligo-nucleotide probe, and immunoperoxidase staining with a monoclonal antibody against LMP1. In situ hybridization of the null cell line HLN-STL-C established from an adult T-cell leukemia patient showed that 100% of the cells contained both EBERs and LMP1 mRNA and about 0.1% of the cells contained gp350/220 mRNA, indicating that a few of the null cells which carried the EBV genome spontaneously entered the late EBV replication cycle.