It is largely unknown whether hepatitis B virus (HBV) sequence variation during chronic infection hampers HBV immune recognition or the antiviral effect of cytokines on HBV production. Here we have analyzed which region of the HBV genome changes most drastically during an interferon-alpha (IFNalpha)-stimulated immune response. In addition, we have investigated whether the mutations affect viral replication, gene expression, and immune recognition of the mutant viral proteins. The study was performed with full-length HBV genomes taken longitudinally from a patient who transiently cleared HBV and seroconverted to anti-HBe during a long-term IFNalpha treatment. We found a replacement of the predominant virus population during IFNalpha therapy The virus populations differed mainly by a cluster of nucleotide changes in the C-gene and a pre-S2 deletion. Most of the newly emerging mutations localized within core/HBe B-cell epitopes, changed HBe antigenicity toward mono- and polyclonal antibodies, and also influenced the reactivity of the anti-HBc/e antibodies of the patient. All genomes tested expressed less HBeAg than wild-type HBV, while replication and IFNalpha susceptibility were similar. These data indicate that IFNalpha therapy can lead to the emergence of HBV variants with mutations mainly affecting recognition of the core/HBe proteins by antibodies. Taken together, the type of core/HBe-specific B-cell immune response, the sequence of the corresponding epitopes, and the HBe expression level appear to contribute to the decision on viral clearance or persistence.