Sindbis virus 26S RNA is the principal species of virus-specific RNA found in the infected cell; it is derived from a one third segment of virion 42S RNA. When translated in cell-free extracts from mouse ascites cells or rabbit reticulocytes, 26S RNA directed the synthesis primarily of the 33,000 dalton virus capsid protein, and the protein products were in the form of free peptides rather than peptidyl-tRNA. In contrast, the polypeptides synthesized in either extract in response to Sindbis virus 42S RNA were heterogeneous, ranging in molecular weight from 33,000 to 190,000, and were largely in the form of peptidyl-tRNA. The number of independent initiation sites on the 26S and 42S RNAs was determined by analyzing a tryptic digest of reaction products labeled with yeast N-formyl-35S-methionyl-tRNAFmet. The 26S RNA appeared to contain a single initiation site, and this site could also be found in varying amounts in different preparations of 42S RNA. However, a second initiation site, distinct from that of 26S RNA, was the major site in 42S virion RNA. These results suggest that 42S virion RNA contains two potential sites for initiation of protein synthesis. Only one of these may be active, however, and it is postulated that the second site functions primarily, if not exclusively, in the subgenomic 26S RNA species. In this regard, Sindbis virus 42S RNA may represent a novel form of a eucaryotic messenger RNA.