The TMV-encoded 30-kDa protein has been implicated in the cell-to-cell transport of TMV in the infected plant. The polyethylene glycol-mediated inoculation of tobacco protoplasts with TMV particles and TMV RNA was used to compare the time courses of the viral 30-kDa protein synthesis in vivo. Upon infection of protoplasts with TMV RNA, the synthesis of the viral 30-kDa protein starts after 4 to 6 hr, has its maximum after 8 to 10 hr, and decreases. After inoculation of protoplasts with TMV, however, the start of the viral 30-kDa protein synthesis and its maximum are delayed by 2 hr, followed by the same decrease. We show that actinomycin D dramatically stimulates the synthesis of the 30-kDa protein by up to 2 orders of magnitude, whereas the synthesis of the viral 126 kDa, the 183 kDa, and the coat protein is increased only by a factor of 2. Surprisingly, actinomycin V is twice as active as actinomycin D, whereas actinomycin I is nearly inactive. The specific stimulation of the 30-kDa synthesis by actinomycin D in vivo depends neither on the Nicotiana variety nor on the TMV strain used. Final evidence that the 30-kDa protein is truly TMV-derived is provided by the slightly different electrophoretic mobilities of the 30-kDa proteins encoded by TMV strains vulgare, dahlemense, and U2. The identification of the 30-kDa protein in two-dimensional gels was achieved for the first time by a combination of ionic and nonionic detergents for the solubilization of the 30-kDa protein and by the specific stimulation of its synthesis by actinomycin D. The mechanism of the strong and selective actinomycin effect on the viral 30-kDa protein synthesis in vivo is as yet obscure. Actinomycin does not appear to act directly on viral protein biosynthesis, since it neither stimulates the 30-kDa synthesis upon translation of TMV RNA in vitro nor alters the ratio of the products. Actinomycin may rather act by inhibiting selectively the synthesis of a host factor whose synthesis starts at least 4 hr after TMV infection and which strongly inhibits the expression of the viral 30-kDa transport protein.