The BamHI WYH region of the Epstein-Barr virus (EBV) genome encodes a protein localized to the nucleus of the infected cell, the EBV-determined nuclear antigen EBNA2. We have constructed a series of recombinant vectors that carried the complete EBNA2 gene, or the gene modified so as to contain defined deletions involving presumed exons and regulatory elements of the gene. The recombinant vectors were transfected into COS-1 cells which permit the replication of simian virus 40 origin-containing plasmids to a high copy number, and the transient expression of EBNA2 was analysed. A recombinant plasmid that carried a BglII-NotI subfragment of the BamHI WYH region (nucleotides 44664 to 50628) contained all the information necessary for inducing the expression of a full length EBNA2 polypeptide. Moreover, EBV DNA sequences between nucleotides 45,442 and 48,337 could be deleted without interfering with the ability of the vectors to induce EBNA2. On the other hand the loss of the left one-third of the BglII-NotI fragment completely abolished the EBNA2-inducing capacity of the vector. A rightward promoter consensus sequence in the BamHI W part of the BglII-NotI fragment was functional in COS-1 cells expressing EBNA2 and essential for EBV-specific RNA synthesis. The results indicated that transcription of the EBNA2 gene was initiated in the BamHI W fragment, that the transcript was spliced and that all of EBNA2 was encoded within the continuous long open reading frame in the BamHI Y and H fragments.