We have developed a method for detecting in human plasma a form of prolactin (PRL) with an apparent molecular weight (MW) of approximately 25,000, distinct from traditional human PRL (hPRL) which has a MW of 23,000. The method is based upon the higher MW of the variant and its ability to partially cross-react with antibody to 23K hPRL. It consists of retrieval of the protein from plasma by immunoprecipitation, its separation from 23K hPRL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto nitrocellulose paper by electroblotting, followed by detection with immunostaining and autoradiography. The method is sensitive enough to detect hPRL and its 25K variant in 100 vl of human plasma. All plasma tested so far has shown the presence of the new form. The method has also revealed another protein in human plasma that cross-reacts with a specific antibody to 23K hPRL. This protein may constitute an additional post-translational variant of hPRL. The 25K variant reported here most likely represents the glycosylated form of the hormonerecently detected in ovine and human pituitary glands. The method described can be adapted to detect other plasma components for which an antibody is available