The ER plays an important role in the proliferation and differentiation of lactotrope tumor cells. GH(4) cells were infected with adenoviral vectors (AdL540Q and Ad1-536) to investigate the ability of dominant negative ER mutants to affect the regulation of gene expression and cell growth by endogenous ER. The dominant negative mutants suppressed estradiol stimulation of an estrogen-responsive reporter gene and the PRL promoter in these cells. AdL540Q or Ad1--536 infection also inhibited GH(4) cell growth and induced apoptosis, increasing the expression of the proapoptotic Bax protein and decreasing the expression of antiapoptotic Bcl-2. AdwtER-infected cells also showed decreased Bcl-2 protein. E2-induced activation of p38 MAPK, an enzyme that may participate in apoptosis, was observed in cells infected with AdwtER, AdL540Q, and Ad1--536. Consistent with the apoptotic effects in vitro, infection of GH(4) cells with AdL540Q or Ad1--536 inhibited the ability of the cells to form tumors in nude mice. These results indicate that dominant negative ER mutants induce apoptosis of GH(4) cells and suppress tumor formation and development. The delivery of dominant negative ERs by adenoviral vectors may provide an alternative modality for the targeted therapy of pituitary lactotrope adenomas and other estrogen-responsive tumors.