The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA f Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA f Met (retic., E. coli ). Other aminoacyl tRNAs tested including fMet-tRNA f Met (retic., E. coli ), Phe-tRNA ( E. coli ), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA f Met forms the initiation complex Met-tRNA f Met:IF1:GTP (2), and in this ternary complex Met-tRNA f Met is not degraded by the deacylase. E. coli Met-tRNA f Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA f Met is degraded by the deacylase. Prior incubation of f1 with Met-tRNA f Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA f Met (retic.) was pre-incubated with peptide chain initiation factors.