A large short-lived virus-specific nonstructural protein with an apparent molecular weight of about 250000 (nsp250) has been isolated from cells infected with the temperature-sensitive mutants ts-4 and ts-6 of the Semliki Forest virus. nsp250 contained all peptides characteristic of the two previously identified nonstructural precursor proteins, nsp155 and nsp135, as revealed by limited proteolysis with Staphylococcus aureus V8 protease. Thus nsp250 is probably the translational product of the 5' two-thirds of the 42-S RNA genome which codes for the virus-specific nonstructural proteins. A second viral nonstructural precursor protein, nsp220, was also characterized by peptide mapping. This protein contained all the peptides of nsp155, and several but not all of the peptides of nsp135. Some peptides were demonstrated which possibly are derived from ns60, the only nonstructural protein not yet isolated. Small amounts of proteins with identical mobility to nsp250 and nsp220 were synthesized at 38 degrees C in micrococcal-nuclease-treated rabbit reticulocyte lysate in response to virion 42-S RNA from the ts-6 mutant. The product of the wild-type 42-S RNA in vitro contained, in addition to nsp220 and nsp155, polypeptides which comigrated with ns86, ns72 and ns70, indicating processing of the translational product. The authenticity of nsp220, nsp155 and ns70 synthesized in vitro was confirmed by limited proteolysis with V8 protease.