ABSTRACT A simple and rapid homologous radioimmunoassay of human prolactin (hPRL), by the use of 66% ethanol containing 6.6% ammonium acetate for the separation of free and bound hormones, has been established. PRL levels showed marked individual and daily variations as well as a circadian periodicity in both men and women. Levels of plasma PRL rose with puberty and decreased during post-menopause and in elderly men (ages 60–73 years). There was no marked change in plasma PRL levels at midcycle; however, in some subjects, luteal levels were slightly higher than follicular levels. Administration of oestradiol-17β caused marked increase in plasma PRL levels during menstrual cycle. During pregnancy, the plasma PRL levels rose as high as 200 ng/ml and decreased within a week or two post-partum. During lactation, the levels were higher and increased with suckling. In a patient with habitual abortion, the plasma PRL level showed a sudden drop prior to abortion. Administration of LH-RH had no effect on the secretion of PRL in human subjects.