An Escherichia coli cell-free translational system, deprived of initiation factor IF-3, has been used to study the role of the factor in protein synthesis. In this system, 30-S ribosomal subunits are preincubated together with MS2 phage RNA in a small volume in the presence of 10 mM Mg(Ac)2; the missing components required for protein synthesis are then added and assembly of elongating ribosomes is allowed to occur. This stepwise assembly process permits formation of functional complexes which can carry out protein synthesis in the complete absence of IF-3. The translational products, obtained in the absence of IF-3, have been analysed and shown to be similar to those synthesized in the presence of the factor. The main product observed is the phage coat protein.