Full-length DNA copies of RNAs 1, 2, and 3 of CMV Y strain (CMV-Y) were cloned downstream of modified phage T7 promoter sequences to obtain infectious RNA transcripts. The small number of extra nonviral nucleotides at the 5' ends considerably decreased the specific infectivity of the transcripts of RNAs 1 and 2 but did not affect that of the RNA3 transcripts. Using the most infective transcripts, up to 45% of tobacco protoplasts could be infected. Various cDNA mutants were constructed from the full-length RNA3 cDNA to give RNA transcripts having deletions in the coding region of the 3a protein or the coat protein. These mutants replicated in tobacco protoplasts but did not produce systemic symptoms on tobacco when inoculated together with transcripts of RNAs 1 and 2. One of the mutants having a small in-frame deletion near the N-terminal region of the coat protein produced local lesions on cowpea and local chlorotic spots on the inoculated leaves of tobacco. These results suggest that both the 3a protein and the coat protein are involved in virus transport, and that viral assembly is associated with long-distance movement of CMV.