Summary The results reported here indicate that the ribosomal decoding site for initiator tRNA exhibits certain features which differentiate it clearly from other aminoacyl-tRNA binding sites: o (1) Tetracyclin, unless used at very high concentrations, does not prevent binding of initiator tRNA, suggesting that this tRNA does not enter the amino-acyl-site. (2) Initiator tRNA reacts with puromycin during the first minutes of its binding, whether at low temperature (13°C) or at 25°C. (3) «Heat activation of 70 S ribosomes or a preincubation treatment in the absence of other ligands, can greatly increase both the rate and extent of F-met-tRNA binding at 13° or 25°C. (4) Streptomycin releases F-met-tRNA as free tRNA from the initiation complex and it does so when initiator tRNA is in the P. site. Yet, no such antibiotic induced release is observable (a) when binding at 25°C proceeds for a very limited period; (b) when the binding reaction occurs at 13°C regardless of the binding extent thus achieved; (c) on 70 S ribosomes at temperatures above 25°C if GMP-PCP (a GTP analog) is used or (d) on 30 S subunits. It is suggested that under these conditions proper adjustement of F-met-tRNA to the P site might not occur and that the conformation of the complex thus achieved is insensitive to streptomycin. Appearance of this particular conformation might normally precede the correct F-met-tRNA positioning at the P site.