The replicase proteins p33 and p92 of Cymbidium ringspot virus (CymRSV) were found to support the replication of defective interfering (DI) RNA in Saccharomyces cerevisiae cells. Two yeast strains were used, differing in the biogenesis of peroxisomes, the organelles supplying the membranous vesicular environment in which CymRSV RNA replication takes place in infected plant cells. Double-labelled immunofluorescence showed that both p33 and p92 replicase proteins localized to peroxisomes, independently of one another and of the presence of the replication template. It is suggested that these proteins are sorted initially from the cytosol to the endoplasmic reticulum and then to peroxisomes. However, only the expression of p33, but not p92, increased the number of peroxisomes and induced membrane proliferation. DI RNA replication occurred in yeast cells, as demonstrated by the presence of monomers and dimers of positive and negative polarities. Labelling with BrUTP showed that peroxisomes were the sites of nascent viral synthesis, whereas in situ hybridization indicated that DI RNA progeny were diffused throughout the cytoplasm. DI RNA replication also took place in yeast cells devoid of peroxisomes. It is suggested that replication in these cells was targeted to the endoplasmic reticulum.