Abstract A stable mRNA-dependent cell-free translation system from Saccharomyces cerevisiae, prepared by a modification of the method of Hofbauer et al. [Eur. J. Biochem. 122 (1982) 199–203] was active in translation of exogenous homologous and heterologous mRNAs. Optimal translational activity required the addition of polyamines and yeast tRNA. The m transcript of the M segment of double-stranded RNA, synthesized in vitro using the killer virus-associated RNA polymerase, directed the synthesis of preprotoxin polypeptide (M-p32), which was immunologically identified using antitoxin antibody. Sindbis virus capsid protein and rabbit globin were also translated from their mRNAs. Translation was inhibited by puromycin, sparsomycin and anisomycin. Analogues of the 5'-terminal caps present on most eukaryotic mRNA molecules inhibited translation of added mRNAs, including capped mRNAs and the uncapped killer virus mRNA.