Immunoassayable TRH in human ejaculate was eluted from a gel column in a form with a molecular weight larger than that of the native peptide. With reverse-phase high-performance liquid chromatography (HPLC) the same activity co-eluted with standard TRH. Incubation of ejaculates at room temperature for 8 h was associated with a time-related increase in the total immunoassayable TRH. Analysis by HPLC of ejaculates after 12 h of incubation at room temperature indicated that, whereas the levels of the peptide co-eluting with native TRH declined with time, there was a concomitant increase in the concentration of a molecular species which also cross-reacted with the TRH antiserum, but which was more hydrophobic. The latter species is presumably identical to the tetrapeptide recently described by others and which may arise from the proteolytic degradation of secretory macromolecules. Although immunological activity was present in all six fractions of split ejaculates, the bulk of the peptide was associated with the later portions, implying a major vesicular contribution. However, secretions isolated from surgical preparations of the seminal vesicles contained undetectable levels of peptide, suggesting that the ejaculation process may represent a stimulus for its appearance in the semen. This study is further support for a local involvement of TRH in male reproductive function.