In women, the concentration of prolactin in the plasma increases in response to nipple stimulation. This response has led to the assumption that prolactin influences the rate of milk synthesis. To investigate this hypothesis we have measured 24 h milk production, the short-term (between breastfeeds) rates of milk synthesis and the concentration of prolactin in the blood and breastmilk, from 1 to 6 months of lactation in eleven women. Over the long term, the 24 h milk production remained constant (means +/- S.E.M.): 708 +/- 54.7 g/24 h (n = 11) and 742 +/- 79.4 g/24 h (n = 9) at 1 and 6 months, respectively. The average short-term rate of milk synthesis (calculated from the increase in breast volume between breastfeeds; means +/- S.E.M.) did not change: 23 +/- 3.5 ml/h (n = 23) and 23 +/- 3.4 ml/h (n = 21) at 1 and 6 months, respectively. However, significant variation in the short-term rate of milk synthesis (from < 5.8 to 90 ml/h) was found both between breasts, measured concurrently (coefficient of variation, c.v. = 72%), and within the same breast, measured over consecutive breastfeeds (c.v. = 85%). The basal and suckling-stimulated concentrations of prolactin in the plasma (means +/- S.E.M.) declined from 1 to 6 months (basal, from 119 +/- 93 to 59 +/- 29 micrograms/1; peak, from 286 +/- 109 to 91 +/- 44 micrograms/l). In contrast, the concentration of prolactin in milk was much lower than in plasma, and decreased only slightly from 1 to 6 months of lactation (fore-milk, from 26.4 +/- 10 to 23.3 +/- 9.8 micrograms/l; hind-milk, from 18.9 +/- 5.1 to 13.2 +/- 6.3 micrograms/l). The concentration of prolactin in the milk was related to the degree of fullness of the breast, such that the concentration was highest when the breast was full. We found no relationship between the concentration of prolactin in the plasma and the rate of milk synthesis in either the short or long term. However, the relationship between the concentration of prolactin in milk and the degree of fullness of the breast suggests that the internalization of prolactin, after binding to its receptor, may be restricted when the alveolus is distended with milk.