Native interleukin-2 (IL-2) contains three cysteines; two exist in a disulfide bridge (Cys-58 and Cys-105) and the third Cys-125 is a free sulfhydryl. In the presence of 6 M guanidine hydrochloride at alkaline pH, IL-2 is converted into three isomers. Each isomer represents one of the three possible disulfide-linked forms that can be generated from three cysteines. These three isomers were resolved on a C4 reverse-phase HPLC system. The identity of each of the three forms was determined by carboxymethylation of the free cysteines in each isomer with [3H]iodoacetic acid followed by determination of the labelled cysteines by tryptic peptide mapping. Tryptic peptide mapping of the more predominant of the two scrambled peaks showed it to be the Cys-105-S-S-Cys-125 linked form of IL-2. A Ser-125 construction of IL-2, which lacks a free cysteine, did not scramble under these conditions. These experiments demonstrate the utility of reverse-phase HPLC in studies of protein folding and disulfide bond structure.