Prolactin is required from d 70 to 110 of gestation for normal mammary development of gilts. The goal of the present study was to determine the effect of inhibiting prolactin with bromocriptine during specific time windows during the second half of gestation on mammary gland development in gilts. Crossbred primigravid gilts were assigned as controls (n = 12) or received 10 mg of bromocriptine orally three times daily from d 50 to 69 (BR50, n = 12), d 70 to 89 (BR70, n = 12), or d 90 to 109 (BR90, n = 12) of gestation. Jugular blood samples were collected on d 50, 70, 90, and 109 of gestation and assayed for prolactin and estradiol. Gilts were slaughtered on d 109 of gestation and fetuses were counted and weighed. One row of mammary glands was used for dissection of parenchymal and extraparenchymal tissues, and for biochemical analyses. Tissue from the other row was used for measures of prolactin receptor number and affinity. Concentrations of prolactin were decreased markedly (P < 0.001) at the end of each bromocriptine treatment period compared with controls, but there was no overall treatment effect (P > 0.1) on estradiol concentrations. Extraparenchymal tissue weight of the mammary glands was unaffected by treatments (P > 0.1), but weight of parenchymal tissue, total DNA, and total RNA were lower (P < 0.01) in BR90 than control gilts. The percentage of DM in parenchymal tissue was unaffected by treatments (P > 0.1), but percentage of fat was higher and percentage of protein lower (P < 0.01) in BR90 gilts compared with controls. Cell size, as estimated by the protein:DNA ratio, also was lower (P < 0.01) in the BR90 group. Number and affinity of prolactin receptors in parenchymal tissue were not significantly altered by treatments. In conclusion, there is a specific time period in the second half of gestation, from 90 to 109 d, during which prolactin is essential for normal mammary parenchymal tissue development.