The release and synthesis of prolactin were studied in incubated adenohypophyses from ovariectomized rats. After a 4 h incubation period the prolactin concentration in the medium markedly increased whereas that in the gland was reduced. However, the concentration of prolactin in the system, tissue plus medium, after 4 h was almost twice as much as that present at the beginning of incubation indicating spontaneous synthesis. This spontaneous release and synthesis of prolactin was greatly increased in incubated glands from ovariectomized oestrogen-treated rats. Oestradiol benzoate was injected in doses of 2.5, 5.0 or 10.0 microgram/rat 2 or 24 h before killing the animals. Lower effects were obtained in glands from 2 h-oestradiol-pre-treated rats than from 24 h-oestradiol-primed rats. Oestradiol-17beta (55, 166, 500 and 1500 ng/ml) added to the incubation medium also enhanced the release and synthesis of prolactin and the effect was more marked in glands from oestrogen injected rats than in those of non-treated animals. The increase was dose-related although the higher doses were less effective. These results provide further evidence of the effect of oestrogen on the release and synthesis of prolactin by a direct action on the pituitary gland. They also show that oestradiol pre-treatment in vivo increase the response of the prolactin cells towards oestradiol in vitro.