The ribosomal peptidyl transferase is a biologically essential catalyst responsible for protein synthesis. The reaction is expected to proceed through a transition state approaching tetrahedral geometry with a specific chirality. To establish that stereospecificity, we synthesized two diastereomers of a transition state inhibitor with mimics for each of the four ligands around the reactive chiral center. Preferential binding of the inhibitor that mimics a transition state with S chirality establishes the spatial position of the nascent peptide and the oxyanion and places the amine near the critical A76 2'-OH group on the P-site tRNA. Another inhibitor series with 2'-NH 2 and 2'-SH substitutions at the critical 2'-OH group was used to test the neutrality of the 2'-OH group as predicted if the hydroxyl functions as a proton shuttle in the transition state. The lack of significant pH-dependent binding by these inhibitors argues that the 2'-OH group remains neutral in the transition state. Both of these observations are consistent with a proton shuttle mechanism for the peptidyl transferase reaction.