Several molecular forms of immunoreactive human (h) PRL with differences in biological activities have been described. To investigate these differences in PRL secretion, we have characterized the molecular forms of circulating PRL in postpartum women during breastfeeding (n = 5) and sleep (n = 5) and in eumenorrheic women with galactorrhea (n = 5) after TRH stimulation (n = 4), metaclopramide infusion (n = 5), and morphine administration (n = 2). All provocative testing in eumenorrheic women were carried out during the early follicular phase of the menstrual cycle (days 1-5). PRL variants were identified and semiquantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography. Mean basal levels of PRL were significantly elevated in nursing women (P less than 0.01) compared to those in nonlactating postpartum women and women with galactorrhea. The relative proportions of PRL variants [glycosylated PRL with an apparent mol wt of 25K and a nonglycosylated species (hPRL) with an apparent mol wt of 23 K] were similar for all three groups. A third immunoreactive PRL variant with an apparent mol wt of 21K was identified in nonlactating postpartum women. With breastfeeding or sleep, PRL concentrations increased more than 4-fold and were accompanied by a shift toward the lower mol wt, nonglycosylated species. In response to TRH (200 micrograms, iv), PRL levels increased by 493 +/- 86% (mean +/- SE) over basal levels. This acute release of PRL was positively correlated (r = 0.06074; P less than 0.001) with a significant increase in secretion of the hPRL species (P less than 0.05). During metaclopramide infusion, a similar increment in hPRL was observed during acute PRL release. Administration of the opiate agonist morphine was associated with a small increment in PRL release and did not alter the proportions of PRL variants in the circulation. Because glycosylation is a posttranslational step in the processing of PRL, our studies suggest that the acute release of PRL in response to physiological and pharmacological secretagogues may reflect the output of more recently synthesized PRL from the pituitary. The physiological role of the 21K PRL species remains to be determined.