We have identified proteins involved in the peptidyl-tRNA-binding site of rat liver ribosomes, using an affinity label designed specifically to probe the P-site in eukaryotic peptidyl transferase. The label is a 3'-terminal pentanucleotide fragment of N-acetylleucyl-tRNA in which mercury atoms have been added at the C-5 position of the three cytosine residues. This mercurated fragment can bind to rat liver peptidyl transferase and function as a donor of N-acetylleucine to puromycin. Concomitant with this binding, the mercury atoms present in the fragment can form a covalent linkage with a small number of ribosomal proteins. The major proteins labeled by this reagent are L5 and L36A. Four protein spots are found labeled to a lesser extent: L10, L7/7a, L3/4 and L25/31. Each of these proteins, therefore, is implicated in the binding of the 3'-terminus of peptidyl-tRNA. The results presented here are correlated with other investigations of the structure-function aspects of rat liver peptidyl transferase. Using these data, we have constructed a model for the arrangement of proteins within this active site.