Pituitary cells have been used for the study of hormone synthesis, secretion, and regulation. However, the lack of human cell lines of pituitary origin has made such studies in humans very difficult. Activin, a member of the transforming growth factor-beta cytokine family, is secreted by the pituitary and serves, in addition to regulating hormone biosynthesis, as a regulator of cell growth and differentiation. In the human pituitary, folliculo-stellate cells secrete an activin-binding and -neutralizing protein, follistatin. However, the role of these cells in the autocrine/paracrine regulatory mechanisms of activin is poorly understood. We describe a human pituitary-derived folliculostellate cell line, designated PDFS, that was developed spontaneously from a clinically nonfunctioning pituitary macroadenoma. PDFS cells showed an epithelial-like morphology with long cytoplasmic processes. Electron microscopy revealed frequent intercellular junctions, including desmosomes, and cytogenetic analysis showed clonal characteristics with chromosomal abnormalities. These cells express vimentin and the nervous tissue-specific S-100 protein, specific markers of folliculostellate cells in the anterior pituitary, but no secretory pituitary cell markers. PDFS cells formed large colonies in an anchorage-independent transformation assay. They express follistatin and activin A and have an intact activin intracellular signaling pathway as determined by reporter assays. Therefore, this human cell line provides a useful model for studying the regulation of cell growth and cytokine production by factors endogenously produced in pituitary folliculostellate cells.