We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase.