Mammary arteriovenous differences of prolactin concentration and net mammary uptake of prolactin from blood were quantified near parturition in 9 dairy cows. Six cows were milked once daily for at least 6 d before parturition, and prepartum lactogenesis occurred in 3 of 6 cows. Prepartum milking 2 or more d before parturition abruptly increased secretion of prolactin into blood, but milkings within 1 d before or after parturition did not increase prolactin secretion. Concentrations of prolactin in whole milk sampled over 8 d before parturition (64.5 ng/ml) were substantially greater than those occurring several days after parturition (19 ng/ml). Milk prolactin concentrations were unaffected by the successful induction of prepartum lactogenesis, which greatly increased prepartum yields of milk (2 to 8 kg/milking). Therefore, the alveolar lumenal content of prolactin was greatest in pregnant cows with prepartum lactogenesis. This enhanced content of intraalveolar prolactin before parturition was associated with an absence of mammary uptake of prolactin immediately prior to ejection of the prolactin-containing milk from the alveoli. However, prolactin uptake was quickly restored to about 2 micrograms/min per half udder shortly after milk ejection. During the prepartum period, an enhanced intraalveolar reservoir of 200 to 400 micrograms prolactin, due to induction of prepartum lactogenesis, appears to saturate temporarily all putative sites for uptake of prolactin from blood.