Assembly of replication-competent hepadnavirus nucleocapsids requires interaction of core protein, polymerase and encapsidation signal (epsilon) with viral pregenomic RNA. The N-terminal portion (aa 1-149) of the core protein is able to self-assemble into nucleocapsids, whereas the C-terminal portion (aa 150-183) is known to interact with pregenomic RNA. In this study, two hepatitis B virus (HBV) core mutants (C144Arg and C144Lys) in which the C-terminal SPRRR (Ser-Pro-Arg-Arg-Arg) motif was replaced by a stretch of arginine or lysine residues were generated to test their role in pregenome encapsidation and virus maturation. Mutant or wild-type core-expression plasmids were co-transfected with a core-negative plasmid into human hepatoma HuH-7 cells to compare trans-complementation efficiency for virus replication. Both low- and high-density capsids were present in -the cytoplasm and culture medium of HuH-7 cells in all transfections. Nucleocapsids formed by C144Arg and C144Lys, however, lost the endogenous polymerase activity to repair HBV DNA. Furthermore, in co-transfection of pHBVC144Arg or pHBVC144Lys with a plasmid which produces replication-competent nucleocapsids, the HBV DNA repairing signal was reduced 40- to 80-fold. This is probably due to formation of mosaic particles of wild-type and mutant cores. Results indicated that the SPRRR motif at the core protein C terminus is important for HBV DNA replication and maturation. Additionally, triple-plasmid transfection experiments showed that nucleocapsids containing various amounts of C144Arg and wild-type core proteins exhibited a bias in selecting a shorter pregenome for encapsidation and DNA replication. It is therefore suggested that unknown factors are also involved in HBV pregenome packaging.