Mono-(125)I-(Tyr A19)-insulin (Mono*A19) was prepared by iodinating MC porcine insulin with (125)I in acid medium using iodate (as oxidizing agent), followed by anion-exchange chromatography. Mono-(125)I-(Tyr A14)-insulin (Mono*A14) was prepared by iodinating MC porcine insulin with (125)I, using H(2)O(2)/lactoperoxidase at neutral pH, followed by anion-exchange chromatography. The specific radioactivities were in the ranges of 120-200 and 220-300 mCi/mg for Mono*A19 and Mono*A14, respectively. Analyses of the intramolecular distributions of (125)I demonstrated that the preparations were 97-98% radiochemically pure. In both preparations, 98-99% of the radioactivity was capable of binding to insulin antibodies for up to 6 months of storage of the tracers. The IRI concentration decreased with the duration of storage. The greatest observed fall in IRI concentration was 70%. The ime course could be explained by the assumption that the disintegration of a (125)I-nucleus destroys the immunoreactivity of the insulin molecule in which the decay occurs.