Abstract The synthesis of two classes of late T7 proteins appears to be directed by several polycistronic messenger RNAs. To understand how they are translated in appropriate order during phage infection, we have isolated late mRNA from T7-infected cells and mRNA that was synthesized in vitro with the T7 RNA polymerase and T7 DNA. Using a purified cell-free system in which ribosomal interaction with the message is restricted to the authentic phage initiation sites, we find that late T7 mRNA directs the synthesis of two initiation dipeptides, formylmethionine-alanine and formylmethionine-serine. These dipeptides account for 80% of the incorporated fMet and are synthesized in systems derived from both uninfected and infected female Escherichia coli cells. However, ribosomes isolated from T7-infected female cells apparently contain mRNA corresponding to these same T7 initiation regions since they also direct the endogenous synthesis of formylmethionine-alanine and formylmethionine-serine. This capability increases as a function of time after infection. In contrast, neither uninfected host ribosomes, ribosomes from T7 am 23 (gene 1 mutant lacking T7 RNA polymerase) infected cells, ribosomes from T7-infected male host cells nor ribosomes from cells infected with Qβ RNA phage direct the synthesis of these or other formylmethionine-dipeptides. It is concluded that the ribosomes after T7 infection seem to have a high affinity for the late viral message and that the regions coding for these formylmethionine-dipeptides may represent preferred sites for their entrance on to the message.