Turkey ovomucoid third domain, OMTKY3, is a potent protein inhibitor of most serine proteinases that prefer a neutral residue at the P1 position. It has been a target of increasingly intense scrutiny by site-directed mutagenesis in an attempt to elucidate the sequence-to-reactivity algorithm of the inhibitor. Here we report the total chemical synthesis of (6-56)OMTKY3 using both the stepwise solid phase peptide synthesis (SPPS) technique and the native chemical ligation approach. After refolding and affinity purification, the resultant products were characterized by electrospray ionization mass spectrometry, analytical ion exchange chromatography, measurement of association equilibrium constants with six different serine proteinases, and thermal denaturation studies. The two synthetic proteins were found to be functionally as well as structurally identical to their recombinant counterpart. Moreover, the native chemical ligation and stepwise SPPS techniques gave rise to comparable yields and similar product quality. The native chemical ligation strategy used here presents highly efficient synthetic access to novel analogs of OMTKY3 that can be used for understanding the molecular basis of enzyme-inhibitor recognition.