Lamivudine treatment of individuals with chronic HBV infection leads to a rapid decline of hepatitis B virus (HBV) serum DNA. Because HBV replication quickly reaches pretreatment values following cessation of the drug, we addressed the question of whether changes during therapy in composition and amount of discernible circulating viral DNA and RNA might provide an explanation for this phenomenon. Nucleic acids were extracted from serial serum samples of two chronically infected patients. The first patient was treated with lamivudine for 14 weeks, whereas the second one, who displayed an HBV virus with a core gene mutation, received lamivudine for 10 weeks. Three sequence segments of the HBV genome synthesized successively during replication, namely, X, C, and X-preC, were analyzed via competitive polymerase chain reaction (PCR) and reverse transcriptase (RT)/PCR. HBV transcripts were also analyzed for differential polyadenylation. At the start of treatment, identical DNA copy numbers (10(9)/mL) were found for all three segments in the first patient. C segment DNA displayed the expected rapid decline. X-preC, a target contiguous only on plus-strand DNA, behaved similarly. In contrast, the X segment DNA copy numbers showed a less pronounced decrease, remaining at higher values (10(7)/mL) than the C and X-preC segments (both about 2 x 10(5)/mL) at the end of therapy. X segment RNA displayed a persisting copy number of about 10(7)/mL, whereas C and X-preC RNA decreased to about 10(5) copies/mL. Polyadenylated HBV RNA, both full-length and truncated, initially persisted at 10(5) but decreased to 10(4) to 10(3) copies/mL at the end of treatment. As expected, C segment DNA and RNA were not detected in the second patient, whereas X and X-preC segments showed essentially the same pattern as the first patient, although at a slightly lower level. We conclude that: (1) actual numbers of HBV genome equivalents during lamivudine therapy can be assessed only via X segment DNA, because it is reverse transcribed first; (2) lamivudine induces coexistence of DNA and RNA for the C and X segments at similar levels, indicating drug-arrested intermediates of reverse-transcribed HBV DNA minus-strand; and (3) packaged HBV RNA lacks a poly(A) tail, whereas polyadenylated RNA is likely not packaged.