A cDNA clone encoding the coat protein (CP) gene of cucumber mosaic virus (CMV, Y strain), linked to the caulifiower mosaic virus (CaMV) 35S transcript promoter, was introduced into tobacco tissues by either electroporation or Agrobacterium-mediated gene transfer. The inoculated and upper systemic leaves of transgenic tobacco plants expressing the CMV-CP gene were highly protected from infection with CMV, even when the concentration of CMV-virion inoculum was 100 mug/ml. Inoculated leaves, however, often showed greater susceptibility to CMV RNA than to CMV virions. Protoplasts isolated from these transgenic plants also were protected from infection when inoculated with CMV virions but were as susceptible as the control protoplasts when inoculated with CMV RNA and unrelated viruses, such as tobacco mosaic virus and brome mosaic virus (BMV), and their respective RNAs. The results suggest protection from virus infection in transgenic protoplasts expressing the CMV-CP gene was specific to infection by CMV virions, and interference with an early event in the infection process, such as uncoating virus particles, is probably involved in the resistance of transgenic tobacco plants expressing the CMV-CP gene. The observed resistance against CMV RNA in whole plants suggests virus replication may occur in primary infected cells, but the initiation of the replication process in adjacent cells may be inhibited by the resident CMV-CP gene if RNA encapsidation or binding to the CP is required for cell-to-cell and long-distance transport.