The 1H nuclear magnetic resonance spectrum of bovine trypsinogen differs from that of trypsin. The appearance of the high field region of the spectrum is dependent on temperature, pH and the addition of calcium, lanthanides, the basic pancreatic trypsin inhibitor, and H-Ile-Val-OH. In particular, simultaneous addition of trypsin inhibitor and H-Ile-Val-OH to trypsinogen induces the high field spectrum of the latter to resemble closely that of trypsin, and this change is reversible. Resonances of the N-terminal "activation" hexapeptide of trypsinogen are identified by the use of paramagnetic lanthanides and pH titrations. The spectroscopie evidence indicates a flexible "random coil" conformation for this hexapeptide in trypsinogen. Detailed ring current analyses are described, which are based on ten sets of crystal co-ordinates for trypsin and trypsinogen, and six sets for the trypsin inhibitor, together with the Johnson-Bovey ring current equation calibrated previously for this application. These show that the high field signals of the proteinase and its zymogen in both the free form and its complex with trypsin inhibitor arise from about 16 methyl groups found in the five-sixths of the proteinase not involved in the "activation domain". Tentative assignments of the two most high field shifted signals are proposed. Combination with the spectral results shows that trypsinogen activation involves several subtle differences of conformation and flexibility in localized regions of the protein that were previously thought to be similar within the precision of the crystallographic analyses. © 1980 Academic Press Inc. (London) Ltd.