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Cytosol from ascites cells catalyzed the binding of Phe-tRNA to 40S ribosomal subunits, the synthesis of polyphenylalanine at low concentrations of magnesium, and the translation of encephalomyocarditis virus (EMC) RNA by ribosomes reconstituted from subunits. Thus, ascites cytosol contained the mammalian initiation factors M1, M2, and M3; M2 could be removed by precipitation at pH 5. 2. Cytosol from rat liver or from muscle on the other hand contained only M1; the fraction extracted from liver or muscle microsomes with 1 M KCl (salt-wash-fraction) contained M2 as well as M1. There was no translation of EMC RNA even when cytosol from rat muscle or from liver was supplemented with the homologous salt-wash-fraction; thus neither fraction has active M3 for the translation of EMC RNA.