In two natural HBe-minus hepatitis B virus mutants, expression of HBe protein was abrogated by a nonsense mutation at precore codon 28 and a frameshift mutation at codon 29, respectively. Both mutants contained an additional nucleotide substitution(s) which was found by transfection experiments to be required for efficient packaging of pregenomic RNA. The observed mutational profiles were consistent with the presence of a base-paired region of the pregenome encapsidation signal overlapping the HBe-coding sequence. Results obtained with artificial mutants with significant changes in the primary sequence suggested that base pairing is required but insufficient for efficient pregenome packaging. However, the predicted first four base pairs of the stem are dispensable.