To analyze different mutations in regulatory sequence of prolactin (PRL) gene during the formation of 17 beta-estradiol (E2 ) -induced prolactinoma in eutopic and ectopic pituitary of rats.Male Sprague-Dawley rats transplanted with an isologaus pituitary under renal capsule were treated with subcutaneous implantation of an empty or E2-laden silastic capsule. Reverse transcription-polymerase chain reaction was employed to evaluate the expression of PRL mRNA in pituitary glands, and DNA sequencing was used to analyze the mutation in regulatory sequence of PRL gene.After treated with E2 for 120 days, both the eutopic and ectopic pituitaries were three times more heavier than those from control group (P < 0. 01) , and the body weight of rats was decreased to 42. 90% of the control group (P < 0 01 ). The PRL mRNA expressions in glands from the eutopic and ectopic pituitaries 120 days after treated with E2 were much more than those in untreated pituitary glands (P <0. 01). DNA sequencing showed seven mutations in the regulatory sequence of PRL gene in the eutopic pituitaries 120 days after treated with E2 , while the mutation in the ectopic pituitaries was decreased.Prolactinomas can be induced by chronic treatment with E2 in both the eutopic and the ectopic pituitaries transplanted under renal capsule distant from the hypothalamus. Different mechanisms exist in the formation of eutopic and ectopic prolactinomas.