Foot-and-mouth disease virus (FMDV) is a positive-sense, single stranded RNA virus and its replication involves the synthesis of a negative strand intermediate. In the present study, a strand-specific quantitative RT-PCR assay was developed for analysis of FMDV replication. Strand-specific detection of viral positive and negative strand RNA was achieved using a high reverse transcription (RT) temperature (62 degrees C) and a tagged RT primer. In both the positive and negative strand assays, the lowest reliably detectable concentration was 1 x 10(2) copies/microl. The assays developed were successfully used to analyse viral replication in tissues collected from experimentally infected sheep during both acute and persistent infection. The results showed that while replication was observed in all tissues examined during acute infection, active viral replication during persistent infection was only detected in the tonsil. These results are consistent with the current opinion that the tonsil in sheep is the main predilection site for virus persistence. This assay will be used in the future to look further at replication in experimentally infected animals, including the study of individual cell types, and will improve our understanding of FMDV pathogenesis.