Toll-like receptors (TLR) play a key role in innate immunity. To examine the ability of diverse TLRs to modulate hepatitis B virus (HBV) replication, HBV transgenic mice received a single intravenous injection of ligands specific for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. All of the ligands except for TLR2 inhibited HBV replication in the liver noncytopathically within 24 h in a ␣/ interferon-dependent manner. The ability of these TLR ligands to induce antiviral cytokines at the site of HBV replication suggests that TLR activation could represent a powerful and novel therapeutic strategy for the treatment of chronic HBV infection.We have previously shown that hepatitis B virus (HBV)-specific CD8 ϩ cytotoxic T lymphocytes and CD4 ϩ helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting gamma interferon (IFN-␥) when they recognize viral antigen (6,8). This antiviral effect could be also induced in response to IFN-␣/ that was produced in the liver during lymphocytic choriomeningitis virus, murine cytomegalovirus, and adenovirus infections (2, 7). More recently, we have demonstrated that NKT cells, NK cells, and antigenpresenting cells inhibit HBV replication when they are activated by alpha-galactosylceramide (16), interleukin-12 (IL-12) (3), , and an agonistic anti-CD40 antibody injection (17), respectively. Collectively, these results suggest that HBV replication can be controlled by innate immune response if it is activated in the liver.Toll-like receptors (TLRs) are essential for the recognition of invading pathogens and serve as an important link between innate and adaptive immunity. TLRs can discriminate various microbial components, such as triacylated lipopeptides (recognized by TLR1/TLR2 heterodimer) (30), diacylated lipopeptides (recognized by TLR2/TLR6 heterodimer) (23), doublestranded RNA (dsRNA; recognized by TLR3) (1), lipopolysaccharide (LPS; recognized by TLR4) (27), flagellin from bacterial flagella (recognized by TLR5) (11), singlestranded RNA (ssRNA; recognized by TLR7/8) (4, 12), and bacterial DNA containing the unmethylated CpG motif (recognized by TLR9) (13). While the TLRs have been shown to play a crucial role in the innate recognition of bacterial and fungal pathogens, recent studies also suggest the importance of TLRs in antiviral immunity in vivo (14,19,20).Although we have reported that poly(I · C) inhibits HBV replication by inducing IFN-␣/ (21), little is known about the ability of other TLRs to control of HBV. Accumulating evidence suggests that each TLR transduces its signals by distinct but overlapping signaling pathways. For instance, TLR3 and TLR4 appear to signal mainly through a MyD88-independent, TRIF-dependent pathway, while TLR2, TLR5, TLR7, and TLR9 signaling appears to be MyD88 dependent and TRIF independent (14). Thus, to examine the potential antiviral effect of these different TLR signaling pathways, groups of three or more age-, sex-, and serum HBeAg-matched transgenic mice from lineage 1.3.32 (9) were injected intravenousl...