Experiments were performed to determine some of the factors responsible for the increase in the fraction of inorganic 131I which always accompanies the release of 131I-iodinated amino acids from thyroid protein during pancreatin digestion of labeled thyroid homogenates. When H2O2 (8 x 10-5 -8 x 102 [image]) was added to pancreatin digest of 131I-labeled rat thyroids, a large increase in inorganic 131I was observed, while at the same time the proportion of 131I present as iodothyronine greatly decreased. There was only a minor effect on the 131I-iodotyrosine fraction. The deiodinating effect of H2O2 could be greatly inhibited by 8.8 x 10-3 [image] methylmercaptoimidazole (MMI), indicating that an oxidative step must be involved in the deiodination process. These observations suggested that the iodide increase that occurred during pancreatin digestion might also be inhibited by a reducing agent such as MML Various concentrations of MMI were tested for such an effect, and it was observed that both the yield of 131I-iodothyronine and the increase in inorganic 131I were markedly affected by the presence of MMI. Omission of MMI from the digestion medium frequently led to destruction of 50% or more of the 131I-iodothyronine. The maximum protective effect of MMI occurred at a concentration of approximately .02[image], a much higher level than has been used by previous investigators. Digestion under anaerobic conditions, without MMI, yielded results comparable to those obtained with .02 [image] MMI under aerobic conditions. Propylthiouracil (PTU) was no more effective than MMI in preventing the deiodination that occurs during pancreatin digestion, in contrast to its much greater inhibitory effect on peripheral deiodination of thyroxine in intact rats. The presence of .02 [image] MMI or the use of anaerobic conditions during the pancreatin digiition did not completely prevent an increase in inorganic 131I. The increase which occurs under these conditions probably involves some mechanism other than oxidative deiodination.