The association of labeled Semliki Forest virus (SFV) RNAs with cytoplasmic structures was studied by sucrose gradient centrifugation after treatment of the total cytoplasm with Triton X-100. Infected HeLa, CE and BHK cells were analyzed in low and high-salt buffers. Independently of the labeling period, about 20% of the viral 42S and 26S RNAs sedimented at 180–400S with newly formed virus-specific proteins and prelabeled ribosomes. Of this material, 70–95% was released by EDTA indicating that a major fraction of the RNA was associated with ribosomes. Only 42S RNA was detected in the polysomes of a mutant, ts-1, which is deficient in the synthesis of 26S RNA. It is concluded that both 42S and 26S SFV RNAs can function as messeng