Improved methods for isolation, characterization, and quantitation of immunochemically heterogeneous forms of calcitonin (CT) in tissue and plasma must be developed before the biological origins and clinical importance of CT moieties can be elucidated. We are now proposing reversed-phase high-performance liquid chromatography (RP-HPLC) as one possible means of achieving high recovery, high resolution of CT moieties. In this paper we report RP-HPLC analyses of trace amounts of radiolabeled and unlabeled synthetic human and rat CT. We have systematically evaluated our application of RP-HPLC by employing several elution modes, including isocratic and gradient elution, as well as several elution reagents. We determined that high recovery and high resolution were best achieved with alkyl ion-pairing reagents, such as tetrabutylammonium phosphate, pH 7.5, or sodium sulfonyl hexane, pH 3.5. The most sensitive UV detection of trace amounts of CT was achieved with tetrabutylammonium phosphate buffer (TBAP). We recommend for RP-HPLC of CT a C18-bonded silica column and elution with a 20-min linear gradient of methanol-water (20:80 to 80:20, v/v) containing 0.005 M TBAP. Combined with appropriate extraction procedures, such as silica adsorption or immuno-adsorbant chromatography, this paired-ion RP-HPLC method can be an important aid in achieving more accurate and extensive information about CT moieties in biological samples. This method will also allow the rapid, optical detection and quantitation of CT moieties recovered from tissues, and perhaps from plasmas.