In investigations of radiolabeling substance P1–11(SP) with 125I isotope, its [Tyr8]-derivative was subjected to the lactoperoxidase (LPO) technique, whereas the native substance P undecapeptide was coupled with 125I-tyrosin-N-hydroxy-succinimidyl ester to the corresponding 125I-Bolton-Hunter SP derivative. For separation of the desired components from their by-products, labeled [Tyr8]-SP was pre-purified by ion-exchange chromatography (IEC) on carboxymethyl cellulose and subsequently investigated by gradient high performance reversed-phase liquid chromatography (gRP-HPLC) with 0.1 M triethyl-ammonium phosphate in acetonitrile (pH 2.5) on an octadecylsilyl silica gel (RP-18) stationary phase. When the total amount of labeled peptide affording a specific activity of approx. 2 × 106 Ci/M was checked by gRP-HPLC, the LPO technique yields about an equimolar mixture of 125I-[Tyr8]-(Met11)-SP and 125I-[Tyr8]-(Met11O)-SP. However, in contrast, substantial enrichment of 125I-[Tyr8]-(Met11)-SP was observed when more retained IEC fractions were analyzed.