Characteristics of pancreatic TRH-degrading activity were determined using [L-proline-2,3-3H] TRH and [L-histidine-2,5-3H] TRH as tracers and thin-layer chromatography to detect, identify and quantify TRH metabolites following incubation of tritiated TRH with pancreatic homogenates. The apparent Km of pancreatic enzymes was 2.2 10–5M, the V, 45 pmol/min, and the apparent specific activity, 62.3 ±3.45 pmol/min/mg total protein. In conditions of enzyme saturation, the percent of TRH degraded was found to be similar to the sum of degraded products formed (TRH-OH and His-Pro). Based on the chromatographic identification of metabolites, the presence of a deamidase pathway and a non-deamidase pathway in the TRH-degradation process of the pancreas was postulated. To better characterize the corresponding pancreatic enzymes, active site-directed inhibitors were then used and metabolites yielded were compared to those obtained in the same experimental conditions using plasma as enzyme source. The detection of His-Pro diketopiperazine among the metabolites was of especial interest since this biologically active metabolite was also found in the pancreas as an endogenous peptide and reported to be either a TRH degradation product or derived from sources other than just TRH. However, in presence of inhibitors, His-Pro diketopiperazine was only detected using plasma as enzyme source. Nevertheless, a pancreatic contribution to plasma TRH-degrading activity cannot be discard