Anterior pituitaries (AP) from sexually mature rhesus monkeys ( Macaca mulatta) were cultured in medium 199. Two AP from nonstressed female animals contained more prolactin activity in the initial medium change than in subsequent medium changes. Pituitaries from animals hemorrhage-stressed (250 ml blood) prior to removal contained similar prolactin activity for each medium change. Analytical disc electrophoresis did not reveal any stainable protein in the medium beyond the initial change, although adequate prolactin activity was present. In experiment 5 four female AP produced large amounts of prolactin continuously for 30 days. Disc electrophoresis revealed 5 components for the initial medium change. Components 4 and 3 persisted with subsequent medium changes and had Rf values of 0.55 and 0.62, respectively. They were tentatively identified as native and deamidated monkey prolactin. An AP from a lactating monkey produced prolactin continuously for 66 days while in another culture three female monkey AP and one male AP secreted prolactin continuously for 96 days. On a per pituitary basis the AP from the lactating animal produced more prolactin than the AP from virgin animals. A similar comparison between the male and virgin female AP indicated that slightly less prolactin was produced by the male pituitary. An antiserum developed to the proteins in monkey AP 5 culture medium (days 9-30 of culture) neutralized the prolactin activity of the medium while an anti-MGH serum did not.