To determine whether prolactin (PRL) could be conclusively demonstrated in human milk, samples from nine puerperal women were examined by radioimmunoassay techniques. Recovery of 78 +/- 5% (mean +/- SE) PRL added to eight milk samples and mean recoveries of 83, 92, and 92% of PRL after 1, 2, and 3 h incubation at 21 degrees C indicated that quantitative recovery was possible. PRL immunoreactivity was not lost by dialysis for 96 h. Serial dilution of milk samples before and after dialysis gave inhibition curves parallel to PRL standards. Milk PRL concentrations were high for the first 3 days after birth, with a peak mean value of 157 +/- 18 ng/ml (SE) on the third day. Milk PRL levels then fell sharply to a mean of 24 ng/ml 13 days after delivery. Milk PRL concentrations showed a significant negative correlation (P less than 0.001) with milk lactose (r = -0.59) and positive correlations (P less than 0.01) with total milk protein (r = 0.49) and alpha lactalbumin (r = 0.33) estimations. We conclude that i) PRL is a normal constituent of human milk, ii) high concentrations of PRL are present in human milk for the first 3 days postpartum but subsequently fall rapidly, and iii) milk PRL levels correlate significantly with milk lactose, total protein, and alpha-lactalbumin values.