HepG2 cells, known to support the replication and virion formation of hepatitis B virus (HBV), were transfected with a cosmid constructed to contain 12 tandem head-to-tail repeats of the HBV genome for effective HBV genome expression. We detected previously identified RNAs of 3.3, 2.3, and 2.0 kilobases (kb) that code for core antigen, large surface antigen, and middle/major surface antigen, respectively. We also detected four additional RNAs of 2.1, 1.7, 1.1, and 0.7 kb [the lengths exclude the poly(A) tail]. S1 mapping and nucleotide sequencing data showed that the 2.1-kb RNA is a spliced RNA whose 5' and 3' ends are identical to those of the 3.3-kb RNA. The results suggest that the 2.1-kb RNA codes for an altered core antigen lacking the last amino acid, cysteine, and that expression of the 3.3-kb pregenomic RNA is regulated, at least in part, by splicing. The map positions of the 1.7- and 1.1-kb RNAs suggest that they code for the carboxyl-terminal portions of the putative polymerase, whereas the 0.7-kb RNA codes for the X protein.