Semen samples were obtained from 4 healthy males on two occasions. One of the samples was liquefied at room temperature for 20 min while the other was diluted immediately with buffer to suppress proteolysis. A pool of immunoextracted calcitonin from liquefied and diluted samples, respectively, was subjected to gel chromatography in a separate experiment. In lyophilized samples the majority of immunoreactive calcitonin (CT) had an approximate molecular weight (Mr) of 10 kilodaltons (Kd) and immunoreactive PDN-21 was also present in the fractions. In diluted ejaculates CT was composed of larger species as well as the 10 Kd form. PDN-21 was present in fractions containing the 10 Kd form but could not be detected in the peaks of larger molecular weight, probably because of lower sensitivity of the PDN-21 assay compared to the CT assay. Our finding of CT and PDN-21 in the same fractions after immunoextraction with CT antibodies suggests the presence of proforms of CT in human seminal fluid.