The emergence of HBe-minus hepatitis B virus (HBV) mutants, usually through a UAG nonsense mutation at codon 28 of the precore region, helps the virus to survive the anti-HBe immune response of the host. Host and viral factors that predispose to the emergence of such mutants are not well characterized. The fact that the precore region forms a hairpin structure essential for the packaging of viral pregenomic RNA may explain the extremely high prevalence of the UAG mutation at codon 28. It converts a wobble U-G pair in the packaging signal between nucleotide 3 of codon 15 (CCU) and nucleotide 2 of codon 28 (UGG) into a U-A pair. Since genotype A of HBV has a CCC sequence at codon 15, the UAG mutation would, instead, disrupt a C-G pair present in the wild-type virus. This alteration was shown by transfection experiments to greatly compromise the packaging of pregenomic RNA. The implication of this finding was elucidated by molecular epidemiological studies. Genotype A was found to be the most prevalent genotype in the wild-type virus populations in France but was found in only 1 of the 46 isolates of HBe-minus mutants found there. These mutants were contributed chiefly by genotype D, the second most prevalent genotype in France, which is characterized by a CCU sequence at codon 15. The role of the single nucleotide at codon 15 was confirmed by the finding of the single genotype A isolate in which both wild-type and mutant viruses were present. Interestingly, nearly all of the mutants had a codon 15 sequence of CCU instead of the CCC present in the wild-type viruses. Our results suggest that genotype A of HBV rarely circulates as HBe-minus mutants, probably because of a requirement for a simultaneous sequence change at codon 15. These data, together with the virtual absence of genotype A in the Chinese samples examined, may provide some insights into the uneven prevalence of HBe-minus mutants in the world.