Aims: A novel integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay was established for detection of infectious hepatitis A virus (HAV). Methods and Results: The specificity of tagged RT-PCR was assessed using HAV genomic positive-strand RNA extracted from HAV virions as reference. Water samples artificially contaminated with infectious or formalin-inactivated HAV were subjected to integrated cell culture (ICC)/RT-PCR and ICC/strand-specific RT-PCR assays respectively. The tagged RT-PCR had high specificity for HAV negative-strand RNA. By demonstrating the formation of negative-strand RNA replicative intermediate, ICC/strand-specific RT-PCR can distinguish between infectious and non-infectious HAV. The described method detected infectious HAV at inoculation level of 10(0) TCID50 per flask within 4 days. Conclusions: The ICC/strand-specific RT-PCR is a novel, rapid, sensitive and reliable method for detection of infectious HAV. Significance and Impact of the Study: Coupled with a suitable virus concentration and purification system, ICC/strand-specific RT-PCR will provide a novel and rapid method for detection of infectious HAV in clinical, environmental and food samples. This assay may be used as an alternative method to test the effective inactivation of inactivated virus vaccines. It may also be adapted to assess the efficacy of disinfection of HAV and enteric viruses in foods and water.