High-vacuum polymerization of alpha-(amino acid)-N-carboxyanhydrides (NCAs) affords polymers with controlled molecular weights and narrow polydispersities; however, it comprehensive study of the end-group composition of the resulting polypeptides has not yet been performed. This reveals crucial information, as the end-groups are indicative of both the polymerization mechanism (i.e., initiation event) and the termination pathways. To this end, poly(O-benzyl-L-tyrosine) initiated by 1,6-diaminohexane was synthesized and Subsequently characterized by MALDI-TOF MS, NALDI-TOF MS, and C-13 NMR spectroscopy to ascertain the end-group structure. Polymers were prepared by both high-vacuum and glovebox techniques in DMF/THF. Preparation of poly(O-benzyl-L-tyrosine) by high-vacuum techniques yielded a polymer initiated exclusively by the normal amine mechanism, and termination by reaction with DMF was observed. In contrast, polymers prepared in the glovebox were initiated by the normal amine and activated monomer mechanisms, and several termination products arc evident. To our knowledge, this is the First rigorous and comparative analysis of the end-group structure, and it demonstrates the advantage of high-vacuum techniques for polymerization of NCAs for the preparation of well-defined polypeptides with end-group fidelity.