Vaccines against rabbit haemorrhagic disease virus (RHDV) are commercially produced in experimentally infected rabbits. A genetically engineered and manufactured version of the major structural protein of RHDV (VP60) is considered to be an alternative approach for vaccine production. Plants have the potential to become an excellent recombinant production system, but the low expression level and insufficient immunogenic potency of plant-derived VP60 still hamper its practical use. In this study, we analysed the expression of a novel multimeric VP60-based antigen in four different plant species, including Nicotiana tabacum L., Solanum tuberosum L., Brassica napus L. and Pisum sativum L. Significant differences were detected in the expression patterns of the novel fusion antigen cholera toxin B subunit (CTB)::VP60 (ctbvp60(SEKDEL)) at the mRNA and protein levels. Pentameric CTB::VP60 molecules were only detected in N. tabacum and P. sativum, and displayed equal levels of CTB, at approximately 0.01% of total soluble protein (TSP), and traces of detectable VP60. However, strong enhancement of the CTB protein content via self-fertilization was only observed in P. sativum, where it reached up to 0.7% of TSP. In rabbits, a strong decrease in the protective vaccine dose required from 48-400 microg potato-derived VP60 [Castanon, S., Marin, M.S., Martin-Alonso, J.M., Boga, J.A., Casais, R., Humara, J.M., Ordas, R.J. and Parra, F. (1999) Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J. Virol. 73, 4452-4455; Castanon, S., Martin-Alonso, J.M., Marin, M.S., Boga, J.A., Alonso, P., Parra, F. and Ordas, R.J. (2002) The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants. Plant Sci. 162, 87-95] to 0.56-0.28 microg antigenic VP60 (measured with VP60 enzyme-linked immunosorbent assay) of crude CTB::VP60 pea extracts was demonstrated. Rabbits immunized with pea-derived CTB::VP60 showed anti-VP60-specific antibodies, similar to RikaVacc((R))-immunized rabbits, and survived RHDV challenge.