Hypophyseotropic dopamine exerts a tonic inhibitory tone on pituitary lactotrophs by the activation of dopamine D2 receptors (D2R). Ablation of D2R through gene knock-out approaches results in hyperprolactinemia and prolactinomas. This phenotype is more severe and develops more rapidly in female mice. We tested whether the female hypersensitivity is due solely to the loss of D2R inhibitory tone or concomitant stimulation by ovarian factors. C57BL/6J congenic D2R(-/-) mice were ovariectomized at 2 months of age and serum PRL levels were measured serially. Ovariectomy attenuated hyperprolactinemia and after 18 months, D2R(-/-) mice had average pituitary weights of 4 mg, compared with 60 mg in the intact group. 17beta-Estradiol did not restore PRL secretion or pituitary weight. Although the pharmacologic dose of estradiol slightly increased pituitary weight in wild-type and D2R(-/-) mice, it inhibited serum PRL in both intact and ovariectomized females and in castrated males. For comparison, we tested the estradiol response of wild-type 129S6/SvEv mice in the same paradigm and found the expected increase in pituitary weight and serum PRL. Our results demonstrate that the development of hyperprolactinemia and prolactinomas in mice lacking D2R is dependent on ovarian stimulation and likely involves a factor(s) in addition to estrogen. Furthermore, we showed that estradiol-induced proliferation and PRL secretion can be differentially regulated in a strain-specific manner. These findings illustrate the importance of genetic background when analyzing endocrine regulation in mutant mouse models.