Hepatitis B virus (HBV) DNA was cloned from serum of a heart transplant recipient who died from fulminant hepatitis B transmitted by the donor. Restriction enzyme analyses of the clones obtained by conventional cloning yielded six HBV variants: a major species (pF-1) representing 88% and five minor species (pF-2 to pF-6), each representing 2% to 4% of the clones. The complete nucleotide sequence of these six variants revealed that five of the six viral genomes, including pF-1, carried a novel 11 base pair (bp) insertion in the core promoter region as well as an 18 bp and an 108 bp in-frame deletion in the pre-S1 region not present in the donor. One genome was identical to the sequence of the donor. Functional analyses of HBV clones generated by in vitro mutagenesis and cassette exchange showed that the 11 bp insertion is a strong binding site for hepatocyte nuclear factor 1 (HNF-1). In transient transfection experiments, the novel HNF-1 sequence motif was shown to result in enhanced viral replication. Immunohistochemical analyses revealed high levels of cytoplasmic and nuclear hepatitis B core antigen (HBcAg) and only scattered hepatitis B surface antigen (HBsAg) expression in the liver. The data in our immunosuppressed patient showed that HBV variants can rapidly accumulate in severe hepatitis B and suggest that the novel HNF-1 binding site may have contributed to the fulminant clinical course, possibly via enhanced viral replication.