The genomic DNA of hepatitis B virus (HBV) is circular and has only one known transcription termination site. The HBV X protein coding sequence is flanked by this transcription termination site at the 3' end and a promoter element at the 5' end. Transcription initiating from the X promoter and terminating at the termination site would produce a transcript 0.7 kb in length, which we have detected in cell lines that produce HBV particles. Unexpectedly, a 3.9-kb transcript containing two copies of the X gene sequence was also detected in these cell lines. Polymerase chain reaction analysis indicates that this 3.9-kb transcript contains sequences from both upstream and downstream of the termination site. Thus, transcription of this 3.9-kb transcript initiates from the X promoter, reads through the termination site, and terminates the second time it encounters the site. Analysis using an SV40-derived vector indicates that the transcription termination site in the HBV genome is also leaky for X gene transcription when a heterologous promoter initiates the transcription. Based on these results, the mechanism of how the transcription termination of HBV mRNA is regulated is discussed.