The dysfunctional coagulation factor XII (FXII) Locarno was purified from 2 L of the proposita's plasma. Studies to identify the molecular defect responsible for the lack of amidolytic and proteolytic activity of this FXII variant were performed. Amino acid sequence analysis of peptides obtained from FXII Locarno on activation with either trypsin or plasma kallikrein and dextran sulfate showed an amino acid substitution of Arg 353 by Pro. Thereby, the kallikrein cleavage site at Arg 353-Val 354 is lost. Although trypsin-activated FXII Locarno was fully cleaved at Arg 334-Asn 335 and at Arg 343-Leu 344, neither amidolytic nor proteolytic activity was generated. We conclude that proteolytic cleavage at Arg 343 in the absence of cleavage at Arg 353 is not sufficient to expose the enzymatic active site in FXII Locarno.