In vivo and in vitro prolactin (PRL)-synthesizing and PRL·releasing activity of fetal (days 12–22) and early postnatal (days 1–10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18 12-15 % of monodispersed pituitary cells displayed PRL immuno-positivity, but only 3-5% of PRL·positive cells were plaque-forming, i.e. releasedPRL. By the end of gestation 19-25% and on postnatal day 1042-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL·positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathke’s pouch cells have a substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions. This study is the first demonstrating PRL release of individual fetal lactotrophs from day 18 of intra-uterine life onwards and the ontogenetic change in the percentage of functionally active and inactive subpopulations of PRL cells. Further, the observations are in line with the assumption that the dopaminergic inhibition of PRL release may start in early postnatal life.