'Tomato bushy stunt virus' (TBSV) and 'Carnation Italian ringspot virus' (CIRV) are members of the Tombusvirus family of single-stranded, positive-sense RNA plant viruses that cause extensive inward vesiculations to the boundary membranes of peroxisomes and mitochondria, respectively. These altered organelles are referred to as multivesicular bodies (MVBs) and are proposed to be the sites of viral RNA replication. Accordingly, MVBs contain the protein components of the viral membrane-bound replication complex; however, the molecular mechanisms underlying the targeting of these viral proteins to their organelle target membranes and their subsequent participation in MVB biogenesis are not well understood. To investigate the role of Tombusvirus proteins in membrane recruitment and MVB formation, a comprehensive analysis of the subcellular sorting, membrane topology, and molecular targeting signals was performed on the TBSV 33-kDa (p33) and CIRV 36-kDa (p36) replication proteins. These analyses revealed that nascent p33 sorts initially from the cytosol to peroxisomes where it is orientated with both its amino and carboxy termini facing the cytosol. The sorting of p33 to peroxisomes is mediated by three peroxisomal targeting elements that resemble targeting signals in resident peroxisomal membrane proteins, including clusters of charged amino acid residues and the protein's two hydrophobic membrane-spanning domains. Additional analysis revealed that p33 sorts also from peroxisomes to a specific subdomain of the endoplasmic reticulum (ER) referred to as peroxisomal ER (pER); the proposed site of nascent peroxisome formation. Peroxisome-to-pER sorting of p33 is mediated by a targeting signal that resembles an arginine-based motif responsible for vesicle-mediated retrieval of resident ER proteins from post-ER compartments. In contrast, p36 sorts to mitochondria and is orientated in mitochondrial outer membranes with its amino and carboxy termini facing the cytosol. Sorting of p36 to mitochondria is mediated by an internal targeting signal composed of the protein's two hydrophobic membrane-spanning domains and the intervening hydrophilic loop region. In addition to sorting to mitochondria, p36 appears to sort also to other, unidentified, subcellular compartment(s), suggesting that, similar to p33, p36 sorts to a membrane compartment involved in the normal biogenesis of mitochondria. Overall, these results provide new insight to the Tombusvirus life cycle and demonstrate how viruses can serve as useful tools for studying organelle biogenesis.