UV irradiation of chicken fibroblasts infected with Semliki Forest or Sindbis virus has been used to investigate the mechanism of synthesis of 42S and 26S RNA, the major plus-strand virus-specified RNAs formed during the multiplication of standard virus particles. From an analysis of the kinetics of UV inactivation of the synthesis of these two RNAs, we conclude (i) that 26S RNA is formed by internal transcriptive initiation from a point about two-thirds of the way from the 3′ end of the 42S negative-strand template; (ii) that there exists a population of plus-strand synthesizing complexes whose members are each capable of synthesizing both 42S and 26S RNA; and (iii) that, on a time-averaged basis, each complex in wild-type virus-infected cells contains one virus polymerase mediating 42S RNA synthesis and three mediating 26S RNA synthesis. The RNA phenotypes of 15 RNA−ts mutants of Sindbis virus have been examined after temperature shift to the restrictive temperature. Under these conditions, cells infected with three mutants, N2, N7, and E268, synthesized four to six times as much 42S RNA (relative to 26S RNA) as wild-type virus-infected cells. These studies were extended by examining, in detail, the RNA and polypeptide phenotypes of mutants N2 and E268. These experiments showed that, in N2- and E268-infected cells, one of the virus-specified nonstructural (NS) polypeptides (NS p89; H. Brzeski and S. I. T. Kennedy, J. Virol. 22:420-429, 1977) is thermolabile after shift up to restrictive temperature. This finding, together with the observation that, after shift, the 26S/42S RNA ratio in N2-infected cells changes markedly in favor of 42S RNA synthesis, leads us to conclude that, of the three NS polypeptides, NS p89 modulates 26S RNA synthesis.