Two reverse transcription-PCR (RT-PCR) assays have been developed for the detection and differentiation of rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), two closely related caliciviruses. In order to select highly specific primers, comparative analysis was performed with a large number of RHDV and EBHSV genomic sequences. Regarding these data, primers were selected from similar regions of the VP60 genes to amplify a fragment of 316 nucleotides from the genome of RHDV and a fragment of 265 nucleotides from the genome of EBHSV. In sensitivity studies, as few as 10 copies of cloned viral genomic fragments were detected in each PCR assay, and no cross amplification was observed between the two viruses. The diagnostic value of the assays was confirmed with clinical material by testing fresh and formalin-fixed, paraffin-embedded liver and spleen specimens from a large number of geographically and temporally distant outbreaks. Thus, the two PCR assays provide highly specific and sensitive, novel means of direct detection of the two caliciviruses. In addition, by detecting the viruses in formalin-fixed, paraffin-embedded tissues (PETs), the RT-PCR assays facilitate retrospective virological and epidemiological studies. For example, the identification of EBHSV in PET specimens collected in the 1970s indicates that this virus appeared in the hare populations several years before the first reports of European brown hare syndrome during the 1980s.