Four kinds of cultured clonal cell line were established from estrogen-induced mammotropic pituitary tumor. These clones showed differences of hormone secretion, morphology, and response to estrogen. One clone tentatively designated MtT/E, consisted of spindle-shaped epithelial cells and showed the strongest adhesion to the culture dish, but did not secrete any pituitary hormone. The second cell line, designated MtT/S, secreted only GH, showed very weak contact with the culture dish, and proliferated almost anchorage independently. the MtT/S cells were mainly spherical and formed floating or weakly adherent clusters. Some of them had very long dendrite- or neurite-like cell processes. Electron microscopic examination of the MtT/S cells showed a normal somatotrope-like appearance, i.e. the presence in the cytoplasm of many GH-containing secretory granules, well developed rough endoplasmic reticulum, and Golgi apparatus. Furthermore, these cells secreted GH in response to stimulation with GRF. The third cell line with an ovoid cell appearance tentatively designated MtT/SM, secreted both PRL and GH, and showed anchorage-dependent proliferation. The fourth cell line designated MtT/Se secreted only a small amount of GH. Among these newly established cell lines, MtT/Se was the smallest in size and showed estrogen-dependent proliferation. The many small secretory-like granules present in the cytoplasm of MtT/Se cells showed no immunocytochemically positive reaction for anterior pituitary hormone. MtT/S and MtT/SM cell lines were also sensitive to estrogen.