In vivo evaluation of CD8 T cell effector (cytotoxic T lymphocyte [CTL]) function in peripheral organs such as the liver is currently not possible but would greatly improve our understanding of local immune regulation, because simple determination of antigen-specific CTL numbers does not predict the outcome of immune responses. In particular, measurement of alanine aminotransferase serum levels is not sensitive enough to detect T cell immunity against low numbers of target hepatocytes. We developed a procedure that detects virus-specific effector function of CTLs in the liver after simultaneous adenoviral transfer of reporter and immune target genes into hepatocytes, followed by bioluminescence imaging of reporter genes. Bioluminescence imaging enabled detection of as few as 10,000 infected hepatocytes in vivo, and even more importantly, quantification of antiviral effector function of as few as 50,000 CTLs. Conclusion: Our results provide evidence that low numbers of antigen-specific CTLs are sufficient to control viral gene expression and eliminate viral infection from hepatocytes. The experimental system established here is a highly sensitive method to simultaneously detect viral infection of hepatocytes and to quantify antiviral CTL function in the liver in vivo and will help in characterizing principles of hepatic immune regulation.