The objective of the study was to determine for Australian wild rabbits the specificity of a competitive enzyme-linked immunosorbent assay (cELISA) developed in Italy for detecting antibodies to RHDV. Analysis of 657 sera collected before the arrival of RHDV (pre-RHD) indicated that between 17 and 38% appeared to give false positive results depending on the cut-off criteria used. The finding of pre-RHD sera reacting positively in the cELISA prompted the testing of sera in a cELISA using as antigen smooth forms of RHDV (cELISA-sf) and a solid-phase ELISA (spELISA), both of which detect reactivity to an epitope shared by the lagomorph caliciviruses. Testing of a subset of the pre-RHD sera in the cELISA-sf and the spELISA revealed that 86 and 91%, respectively, were positive. Similar results were obtained for a set of sera collected pre-RHD in the Australian Capital Territory (ACT). Sera collected from wild-stock rabbits born and raised in isolation in an animal house in the ACT were all negative in the cELISA, 6% were positive in the cELISA-sf and 13% in the spELISA. It was concluded that a calicivirus related to RHDV and European brown hare syndrome virus (EBHSV) was present in the rabbit population before the arrival of RHDV, and may still be present. The potential consequences of these findings for epidemiological studies on RHD in Australia are discussed.