Previous studies have suggested that the uptake of prolactin from the blood into the milk may be restricted when the alveolus is distended with milk. Therefore the aim of this study was to determine the relationship between prolactin in milk and milk production by measuring the concentration of prolactin in samples of fore- and hind-milk as well as the volume of milk removed for each breast, at each breastfeed over a 24 h period. The mean (± S.D.) concentration of prolactin in milk for all women (n = 15) over the 24 h period was 18.5 ± 11.6 µg l-1 (fore-milk) and 16.8 ± 12.8 µg l-1 (hind-milk). The variation between women masked small changes within women in the concentration of prolactin in milk over the 24 h period, therefore a prolactin ratio (individual fore- or hind-milk value divided by the mean for all fore- or hind-milk samples collected over a 24 h period) was determined. The concentration of prolactin was highest in milk between 02.01 and 06.00 h (prolactin ratio for fore- to hind-milk, 1.5), and lowest between 10.01 and 18.00 h (prolactin ratio for fore- to hind-milk, 0.8). Furthermore, we observed that the difference in prolactin concentration between the fore- and hind-milk (fore-hind gradient) was greatest between 06.01 and 10.00 h (4 µg l-1). To ensure that this effect was not due to permeability in the paracellular pathway, the concentrations of serum albumin and sodium in milk were measured. No significant (P > 0.05) changes over the 24 h period, or with increasing time since last feed were observed. We therefore concluded that when the breast is most drained of milk (in the late evening), and the rate of milk synthesis is greatest, the fore-hind prolactin gradient in the milk of the following feed (in the early morning) is highest. This is consistent with the observation that prolactin uptake from the blood by the lactocyte is dependent on the fullness of the breast, such that prolactin uptake may be inhibited in full alveoli. Experimental Physiology (2002) 87.2, 207-214.