The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of α1-antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607–613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display-selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgoño CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291–298], we studied the importance of the scaffold, serpins α1-antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACTE8, ACTG9, AATE8 and AATG9 showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8-based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin-like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.