When actinomycin D-treated chick fibroblasts were labeled with (3)H-uridine for varying periods during the log phase of Semliki Forest virus infection, radioactivity was found associated with different cytoplasmic fractions. After a 1-min period of labeling, it appeared in a large cytoplasmic structure which was seen in electron micrographs of infected cells. Sediments of sucrose density gradients of cytoplasmic extracts of these cells also contained these structures. Three forms of viral ribonucleic acid (RNA) were associated with this cytoplasmic structure: a ribonuclease-sensitive 42S form identical to the RNA of the mature virus, a ribonuclease-sensitive 26S form, and a ribonuclease-resistant 20S form. After a 5- to 10-min labeling period, radioactivity was associated with a ribonuclease-sensitive 65S cytoplasmic fraction which contained only the 26S RNA form. Finally, after a 1-hr labeling period, a 140S ribonuclease-resistant particle was the most prominent radioactive structure in the cytoplasm. This particle contained only 42S viral RNA. Negative-contrast electron micrographs of the 140S particle and the virion demonstrated structural differences between them. The base compositions of the 42S and 26S viral RNA forms were not significantly different. The base composition of the 20S form differed significantly from that of the other two viral RNA forms, but the values obtained for the mole fractions of the bases present in the 20S form differed, and depended on the period during the virus growth cycle in which (32)P was present. These results suggested that viral RNA originated in the large cytoplasmic body. The 20S RNA appeared to be a structure engaged in viral RNA replication and the 140S particle appeared to be a virus precursor.