Rat insulin II, unlike rat insulin I and other non-rodent insulins, contains a unique methionine residue at position B29. Reversed-phase HPLC allows for separation of the two rat insulins, with insulin I typically eluting faster than insulin II. An anomalous peak of insulin immunoreactive material was found eluting even faster than insulin I following acid extraction of rat insulin-producing cells. This early peak co-eluted with [Met-OB29]insulin II suggesting that during cell extraction and subsequent purification steps, rat insulin II is subject to selective oxidation at MetB29. Such oxidation of rat proinsulin II affords improved separation from rat proinsulin I compared to the native form.