The replication of hepatitis A virus (HAV) is via a complementary negative-strand RNA. Each negative strand may serve as a template for the synthesis of many positive strands. The aim of this study was to detect the intermediate replicative (negative strand) of HAV in order to monitor its replication in vitro and in vivo. Real-time polymerase chain reaction (PCR) was standardized to detect the intermediate replicative of HAV in cell culture and liver from non-human primates infected experimentally. HAV primers from the 5' non-translated region and VP3 were used in the cDNA synthesis of negative-strand RNA. The negative strand was detected in the infected cell lines and liver by highly strand-specific rTth recombinant Thermus thermophilus DNA polymerase reverse transcription followed by quantitative PCR. The results indicate that the negative-strand HAV RNA can be detected in vivo and in vitro. This model is an approach for assessing the dynamic patterns of replication and should represent a valuable tool for the monitoring of HAV replications in cell cultures and for the evaluation of experimental infections in animal models.