The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and re-oxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2–19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9–21] and [15–27] native ones. The differences occurred in sections 2–7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19–21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9–21, 15–27] arrangement and resulted in a well-conserved (7–28) segment.