125I‐[Nle15]‐gastrin17 prepared by the iodogen method can be separated by reversed‐phase high performance liquid chromatography into two peaks, both of which elute after [Nle15]‐gastrin17. Direct determination of the specific activities of the two derivatives by microbore reversed‐phase HPLC indicated that they were the mono‐ and di‐iodinated species. In contrast the two peaks obtained with [Met15]‐gastrin17 iodinated under the same conditions eluted earlier, relative to the appropriate gastrin17 standard, than the [Nle15]‐gastrin derivatives. Treatment of either peak with 0.75 M dithiothreitol at 56°C or 95°C resulted in progressive conversion to compounds migrating in relative positions similar to the 125I‐[Nle15]‐gastrin17 derivatives. Direct determination of the specific activity of the earlier eluting [Met15]‐gastrin17 derivative before reduction indicated that it was the mono‐iodinated species. It thus appears likely that iodination of [Met15]‐gastrin17 by the iodogen method results predominantly in the formation of mono‐ and di‐125I‐[Met sulphoxide15]‐gastrin17. To avoid problems arising from oxidation of the methionine residue of gastrin during iodination, the use of 125I‐[Nle15]‐gastrin17 in binding studies is therefore recommended.