Abstract. A method is described which permits culture of primary thyroid cells without subculture for at least 100 days. Cultures are maintained without medium changes for the entire period, and concentrated glucose is added to replenish energy supplies at carefully defined intervals. The cells retain morphological and functional differentiation shown by light and electron microscopy, PAS positive histochemistry, iodine uptake and T4 production for at least 100 days. After this time fairly sudden death of the cultures occurs. Possible mechanisms for the effect are postulated. The technique should make it possible to study long-term effects of drugs/radiation on differentiated cultures without the need for continuous subculture.