Hepatitis B virus (HBV) biosynthesis involves the transcription of the 3.5-kb viral pregenomic RNA, followed by its reverse transcription into viral DNA. Consequently, the modulation of viral transcription influences the level of virus production. Nuclear receptors are the only transcription factors known to support viral pregenomic RNA transcription and replication. The coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1alpha) and corepressor small heterodimer partner (SHP) have central roles in regulating energy homeostasis in the liver by modulating the transcriptional activities of nuclear receptors. Therefore, the effect of PGC1alpha and SHP on HBV transcription and replication mediated by nuclear receptors was examined in the context of individual nuclear receptors in nonhepatoma cells and in hepatoma cells. This analysis indicated that viral replication mediated by hepatocyte nuclear factor 4alpha, retinoid X receptor alpha (RXRalpha) plus peroxisome proliferator-activated receptor alpha (PPARalpha), and estrogen-related receptor (ERR) displayed differential sensitivity to PGC1alpha activation and SHP inhibition. The effects of PGC1alpha and SHP on viral biosynthesis in the human hepatoma cell line Huh7 were similar to those observed in the nonhepatoma cells expressing ERRalpha and ERRgamma. This suggests that these nuclear receptors, potentially in combination with RXRalpha plus PPARalpha, may have a major role in governing HBV transcription and replication in this cell line. Additionally, this functional approach may help to distinguish the transcription factors in various liver cells governing viral biosynthesis under a variety of physiologically relevant conditions.