Generation of replicative defective viruses is frequently observed during viral infections. We now report that encapsidation and reverse transcription of spliced viral RNA is an additional mechanism for synthesis of defective viral particles. We have investigated the in vivo synthesis of a spliced hepatitis B virus (HBV) RNA. By using the polymerase chain reaction with different sets of primers on DNA purified from infected livers and the HepG2 HBV cell line, we detected a subgenomic HBV DNA complementary to the spliced viral RNA. Its nucleotide sequence was found to be identical to that previously described for the spliced RNA. This HBV RNA is packaged and reverse transcribed in vivo, the cDNA being incorporated into circulating particles. This finding establishes the synthesis of spliced HBV RNA in vivo and indicates that its reverse transcription can give rise to defective viruses.