Three immunoreactive prolactin molecules have been separated according to size by chromatography and typified by radioimmunoassay. In the circulation immunoreactive prolactin exists in three forms designated as "extra-big", "big" and "small" after gel filtration on Ultrogel AcA 54. Most immunoassayable prolactin occurs in the small form, which corresponds to monomeric prolactin and presents totally 73.8 +/- 8% in normal subjects. This pattern is not significantly different from that obtained from the serum of normal subjects stimulated by thyroid releasing hormone (TRH) or insulin hypoglycaemia and from pregnant women. The response of big and small prolactin was in proportion to the rise in total prolactin and decrease in the proportion of big-big prolactin in normal women. In no instance was a big prolactin response seen in the absence of a total prolactin response in normal or hyperprolactinaemic patients. Essentially different stimuli have the increase in concentration of big prolactin toward the end of the tests.