Previous experiments using gene transfer of plasmids with heterologous promoters identified an Epstein-Barr virus (EBV) gene (BZLF1) whose product (ZEBRA) switches the virus from a latent to a replicative state. We have now studied expression of ZEBRA in lymphoid cells harboring either standard virus or a mixture of standard and defective (heterogeneous [het]) viruses. A high-titer rabbit antiserum to a TrpE-BZLF1 fusion protein was used to identify ZEBRA expressed from standard and het EBV DNA. These ZEBRA proteins could be distinguished from each other on the basis of their electrophoretic mobilities. ZEBRA could not be detected in cells latently infected with standard EBV. However, within 6 h after induction of replication by sodium butyrate, ZEBRA appeared and persisted long thereafter. Synthesis of ZEBRA was insensitive to phosphonoacetic acid or acycloguanosine, behavior characteristic of an early replicative protein. ZEBRA was constitutively expressed in cells containing both defective and standard EBV genomes. ZEBRA was made predominantly from the het genome but also from the standard genome. Control of BZLF1 expression appears to occur at the transcriptional level. No BZLF1-specific transcript was detected in cells containing only standard latent EBV. BZLF1 transcripts could be detected in these cells if virus replication was induced by treatment with butyrate. Cells bearing both standard and het genomes did not require addition of an exogenous inducing agent to transcribe the BZLF1 gene. The experiments suggest that regulation of transcription of the BZLF1 gene is a pivotal event in the control of EBV replication.