The ability to form a “peptide” bond between various forms of Met-tRNA or Phe-tRNA and puromycin has been studied in the reticulocyte cell-free system. When Met-tRNAF, fMet-tRNAF, or N-acetylPhe-tRNA are used as substrate at low Mg++ concentration (3 mM), reticulocyte initiation factors M1 and M2 (M2A + M2B) are required for puromycin-peptide synthesis. In contrast to bacterial systems, this reaction is also stimulated by the elongation factor T1. When Met-tRNAM or Phe-tRNA is used as substrate, there is no M-factor requirement for the puromycin reaction; T1 is absolutely required, and the reaction is stimulated by T2. These studies indicate that reticulocyte factors M1 and M2 may function in part by placing the initiator tRNA into the P site. The detailed mechanism for mammalian initiation, however, may be more complex than that for bacterial systems.