Large amounts of immunoreactive PDN-21 (iPDN -21) were found in human milk in concentrations similar to those of immunoreactive calcitonin (iCT): 187 +/- 73 pM (mean +/- S.D.) vs 210 +/- 83 pM. (n = 17). Calcitonin (CT) was immunoextracted from milk by means of CT antibodies coupled to Sepharose 4B, and the extracts were gel-chromatographed on Sephadex G-75 after treatment with 6 M guanidine HCl. iCT and iPDN-21 in the fractions were determined with radioimmunoassay. The main part of iCT eluted as high molecular weight forms and these fractions also contained iPDN-21. Enzymatic cleavage of immunoextracted milk CT by trypsin demonstrated that iPDN-21 could be split apart from the high molecular weight forms and be recovered at the position of synthetic human PDN-21 on gel chromatography. iCT was eluted in the region of monomeric CT and as larger forms. Since PDN-21 constitutes the carboxyterminal of preprocalcitonin, our results indicate that human milk contains precursors of calcitonin.