HBeAg seroconversion during the natural history of chronic hepatitis B (CHB) is associated with a strong drop in serum HBV DNA levels and a reduction of intrahepatic covalently closed circular DNA (cccDNA) content. Of particular interest is the transition to HBeAg-negative chronic infection (ENCI). ENCI, previously known as inactive carrier state, is characterized by very low or negative viremia and the absence of liver disease. The molecular mechanisms responsible for the transition to ENCI and for the control of viral replication in ENCI are still poorly understood.To identify what step(s) in the viral life cycle are controlled during the transition to ENCI, we quantified cccDNA, pre-genomic RNA (pgRNA), total HBV RNA and DNA replicative intermediates in 68 biopsies from patients in different phases of CHB.HBeAg seroconversion is associated with a reduction of cccDNA amounts as well as transcriptional activity. Silencing of cccDNA is particularly pronounced in ENCI, where there was ∼46 times less pgRNA per cccDNA compared to ENCHB. Furthermore, a subgroup of ENCHB patients is characterized by reduced replication efficiency downstream of pgRNA.Reduction of HBV serum viral load during transition to ENCI seems to be primarily the results of a strong inhibition of cccDNA transcriptional activity which can be maintained in the absence of liver disease.