Hepatitis B virus (HBV) infection is a major cause of liver-related morbidity and mortality. Toll-like receptors (TLRs) have recently been recognized to play an important role in the pathogenesis of chronic hepatitis B (CH-B). Furthermore, manipulation of TLR signalling pathways shows potential as an antiviral therapeutic strategy. Whether hepatocytes themselves possess intact TLR signalling pathways remains controversial. It is critical that cell culture models be developed to allow investigation of the interaction between HBV and the TLR signalling pathways. We have screened three hepatocyte cell lines for the integrity of pro-inflammatory responses and antiviral cytokines following stimulation with interleukin-1 (IL-1) and different TLR ligands. We observed that Huh-7, HepG2 and PH5CH8 cells selectively responded to IL-1 and TLR2 ligands, leading to the activation of NF-κB. In addition, the PH5CH8 cell lines were able to induce type 1 interferon (IFN) via both TLR3 and RIG-I following stimulation with poly I:C, HepG2 cells mounted an IFN response via RIG-I only, whereas Huh-7 cells were unresponsive. We conclude that the hepatocyte cell lines investigated display a repertoire of TLR signalling, albeit limited, suggesting that hepatocytes may themselves play an active role in innate immune responses to viruses such as HBV. Furthermore, particular hepatoma cell lines are suitable for investigating the interaction between HBV and hepatocyte-expressed pattern recognition receptors.