The possible role of Met-tRNAf deacylase in the regulation of protein synthesis in rabbit reticulocyte lysate by the hemin-controlled translational repressor (HCR) or the double-stranded RNA-activated inhibitor (dsI) has been examined. Inhibition of protein synthesis by either HCR or dsI is associated with a marked increase in the steady state level of 48 S initiation complexes, containing a 40 S ribosomal subunit, globin mRNA, and a reduced level of Met-tRNAf, suggesting that the rate of 60 S subunit addition may be inhibited and that subunit-bound Met-tRNAf may become deacylated by Met-tRNAf deacylase. The addition of highly purified Met-tRNAf deacylase to lysate samples incubated with HCR or dsI reduces the [35S]Met-tRNAf labeling of 48 S complexes to even a lower level but has no effect on the high level of [35S]Met-tRNAf associated with 43 S complexes in the plus hemin control. The effect of added deacylase on the labeling of 48 S complexes with [35S]Met-tRNAf can be overcome by adding eIF-5 or a soluble reticulocyte protein that has been termed the reversing factor, but not by the addition of eIF-2. Added deacylase has no effect on the level of mRNA in 48 S complexes or the labeling of these complexes with [35S]fMet-tRNAf. When lysate samples were labeled with Met-tRNAf, purified from wheat germ or yeast, and doubly labeled with 32P at the 5' end and [35S]methionine aminoacylation, HCR reduced the level of 32P and 35S-labeled tRNAMetf in 48 S complexes to a similar degree, suggesting that once it has become deacylated, tRNAMetf dissociates from the 40 S subunit.