Wheat germ supernatant is resolved into 4 fractions, C3.alpha., C3.beta., D, and A, all of which are required in addition to elongation factors EF1 and EF2 for tobacco mosaic virus-RNA (TMV-RNA)-catalyzed amino acid polymerization. Fractions C3.beta. and D function in the binding of Met-tRNAiMet to the 40 S ribosomal subunit forming an unstable 40 S.cntdot.Met-tRNAiMet complex that is detected in sucrose gradients only after fixation with glutaraldehyde. Fraction C3.beta. binds Met-tRNAiMet suggesting that a C3.beta..cntdot.Met-tRNAiMet complex may be an intermediate in the formation of the 40 S complex. GTP is required for the formation of both the C3.beta..cntdot.Met-tRNAiMet and the 40 S.cntdot.Met-tRNAiMet complexes. In either reaction the nucleotide is partially replaceable by guanyl-5''-yl methylene diphosphonate (GMP-P(CH2)P while ATP is inactive. When ATP, C3.alpha., mRNA, and magnesium acetate (final concentration 3.6 mM) are added to an incubation reaction containing the 40 S.cntdot.Met-tRNAiMet complex, a stable 80 S.cntdot.Met-tRNAiMet complex is formed. Radioactive TM virus-RNA binds specifically to the 80 S.cntdot.Met-tRNAiMet complex suggesting that mRNA is a component of this complex. C3.beta. and D, the fractions required in the formation of the 40 S.cntdot.Met-tRNAiMet complex, are also required for formation of the 80 S.cntdot.Met-tRNAiMet complex. In addition, unlabeled Met-tRNAiMet does not compete significantly with performed 40 S.cntdot.Met-tRNAiMet in the 80 S complex-forming reaction. These observations suggest that the 40 S.cntdot.Met-tRNAiMet complex is an intermediate in the 80 S reaction. The 80 S.cntdot.Met-tRNAiMet product of the sequential reaction is reactive with puromycin and on this basis it is tentatively considered to be a functional initiation complex. The additional requirement of Fraction A for amino acid polymerization suggests, however, that the initiation process may be more complex.