Assembly of the enveloped hepatitis B virus (HBV) is initiated by packaging of the RNA pregenome and the viral reverse transcriptase-DNA polymerase into a nucleocapsid. The pregenome is then reverse transcribed into single-stranded minus-polarity DNA, which is subsequently replicated to double-stranded DNA. All replicative intermediates are observable in capsids within infected liver, but only relatively mature nucleocapsids containing partially double stranded DNA are found in secreted virions. This observation suggests that maturation of the genome within the capsid is required for envelopment and secretion. We show that the differential distribution of replicative intermediates between intracellular nucleocapsids and secreted virions is also observable in human hepatoma cells transfected with wild-type HBV genomes. However, nucleocapsids were not enveloped or secreted when they were produced by an HBV genome carrying a missense mutation in the DNA polymerase that eliminates all DNA synthesis. An HBV missense mutant defective in the RNase H activity of the polymerase which allowed minus-strand DNA synthesis but not formation of double-stranded DNA was able to form virion-like particles. These experiments demonstrate that immature nucleocapsids containing pregenomic RNA are incompetent for envelopment and that minus-strand DNA synthesis in the interior lumen of the capsid is coupled to the appearance of a signal on the exterior of the nucleocapsid that is essential for its envelopment.