We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies.