Abstract This report describes the properties of a purified Escherichia coli protein synthetic system which accurately translates the initial codons of the bacteriophage f2 maturation, coat, and RNA synthetase cistrons. In order to assay cistron recognition easily and to accumulate specific intermediates in the translation of a natural message, we have restricted in vitro synthesis to the formation of the initial formyldipeptides corresponding to the three f2 cistrons. This is accomplished by preventing the initial, G factor-requiring translocation reaction through the use of small amounts of monospecific anti-G factor antibody or the antibiotic fusidic acid. The reaction requires initiation-competent ribosomes which are depleted of elongation factors by repeated washings in low ionic strength buffer. Initiation in this system is quite specific. The initial f2 coat dipeptide, N-formylmethionylalanine, is readily converted into the expected coat tripeptide, N-formylmethionylalanylserine, if translocation is permitted. In addition, synthesis of the initial f2 RNA synthetase dipeptide, N-formylmethionylserine, is specifically repressed in the presence of f2 coat protein, indicating that this purified system is sensitive to translational control.