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Isolated tobacco mesophyll protoplasts were inoculated in vitro with tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) either as a mixture or one after another. Fluorescent antibody staining demonstrated that about 80% of protoplasts produced both viruses following mixed inoculation, and 70% after sequential inoculation. Neither synergism nor antagonism was apparent between TMV and CMV in the establishment of infection. Double infection of protoplasts occurred also with potato virus X (PVX) and TMV, or with PVX and CMV. Inoculation with a mixture of TMV, CMV and PVX caused infection of some protoplasts by all three viruses.