One-dimensional nmr exchange spectroscopy was carried out to determine thermodynamic parameters of cyclophilin-induced cis-trans isomerization of succinyl-Ala-Phe-Pro-Phe-p-nitroanilide. Rate measurements were possible at physiological temperatures. The kc/Km of rat cyclophilin was found to be 12.8 (+/- 0.5) s-1 microM-1 at 37 degrees C, intermediate to previously reported values that used a coupled enzyme assay extrapolated to this temperature. Activation energies (delta G not equal to) for the uncatalyzed and catalyzed reaction at 37 degrees C were found to be 19.7 and 17.1 kcal/mol, respectively, and were primarily due to an enthalpic barrier.