Continuous culture of parathyroid cells has proven difficult, regardless from which species the cells are derived. In the present study, we have used a defined serum-free low calcium containing medium to culture human parathyroid cells obtained from patients with parathyroid adenomas due to primary hyperparathyroidism. No fibroblast overgrowth occurred, and the human parathyroid chief cells proliferated until confluent. After the first passage the cells ceased to proliferate, but still retained their functional capacity up to 60 days, demonstrated by Ca 2+ -sensitive changes in the release of parathyroid hormone (PTH) and as adequate cytoplasmic calcium ([Ca 2+ ] i ) responses to changes in ambient calcium as measured by microfluorimetry. Low calcium concentrations enhanced, and vitamin D 3 and retinoic acids (RA) dose-dependently inhibited cell proliferation during the first passage, as determined by [ 3 H]thymidine incorporation, immunohistochemistry for proliferating cell nuclear antigen and cell counting. Signs of differentiation were present as the set-points, defined as the external calcium concentration at which half-maximal stimulation of [Ca 2+ ] i (set-point c ), or half-maximal inhibition of PTH release (set-point p ) occur, were higher in not proliferating compared with proliferating cells in P0. Inhibition of cell proliferation was accompanied by signs of left-shifted set-points, indicating a link between proliferation and differentiation.The results demonstrate that human parathyroid chief cells cultured in a defined serum-free medium can be kept viable for a considerable time, and that signs of differentiation occur after proliferation has ceased. The low calcium stimulated cell proliferation may also be inhibited by vitamin D and RA.