A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.