The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. Novel molecular techniques have opened new possibilities to investigate the organization and the activity of the cccDNA minichromosome in vivo, and recent advances have started to shed light on the complexity of the mechanisms controlling cccDNA function. Nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels in the course of chronic hepatitis B (CH-B) infection may reveal new potential therapeutic targets for anti-HBV drugs and hence assist in the design of strategies aimed at silencing and eventually depleting the cccDNA reservoir.