The differential rate of systemic symptom induction in zucchini squash by the Fny- and Sny-strains of cucumber mosaic virus (CMV) previously was mapped to RNA 1, which encodes a protein (1a) involved in virus replication. Examination of the kinetics of accumulation of the RNAs and the four encoded proteins in the inoculated cotyledons showed that the Fny-CMV-associated products generally appeared earlier than the Sny-CMV-associated products. In the systematically infected leaves, this difference was magnified, with a 2-day delay in the appearance of the Sny-CMV RNAs and encoded proteins. However, both Fny-CMV and Sny-CMV RNAs showed similar kinetics of RNA, 2a, 3a, and coat protein accumulation in protoplasts prepared from zucchini squash cotyledons. These data indicate that the differential rate of systemic symptom development was due to a difference in the rate of movement rather than the rate of replication. This was confirmed by a leaf-detachment assay, which showed a difference in the rate of systemic movement by Fny-CMV vs Sny-CMV, and by leaf-press blot hybridization of the inoculated cotyledons at different days postinoculation, which showed a difference in the rate of cell-to-cell movement by the two strains of CMV. Taken together, these data show that the rates of cell-to-cell and long-distance movement can be regulated by sequences in CMV RNA 1, previously thought to be involved only in virus replication.