We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate. On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M). Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation. We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site.