Dispersed female rat anterior pituitary cells were cultured in Medium 199 containing 20% fetal calf serum for 30 days. Prolactin levels in the culture medium remained relatively constant during this time, ranging from 30-40 ng/10,000 cells seeded/day. The total quantity of prolactin released into the medium was 10-15 times that originally contained in the cells. Morphological integrity of the mammotrophs was maintained. Using velocity sedimentation at unit gravity, cells from untreated, overiectomized or estrogen-primed animals were separted into several fractions, and subsequently cultured for 14 days. Not all mammotrophs secreted the same quantity of hormone during this time. The data suggest that the pituitary of the female rat is composed of a heterogeneous population of mammotrophs, and that their capacity to secrete prolactin in vitro may, in part, be reflected by the previous physiological status of the animal.