An in vitro system of secondary and tertiary cultures of human thyroid epithelial cells (TFECs) in monolayer is described. The function of the cells was evaluated by the second messenger cAMP and the end product thyroglobulin (Tg). The Tg production from the cells was measured in the supernatant by a newly developed enzyme-linked immunosorbent assay. The TFECs in secondary monolayer cultures had preserved the ability to produce Tg and cAMP despite lack of polarization. Furthermore, a preserved ability of TSH-stimulated production of Tg and cAMP in 12-week-old secondary and tertiary cultures was found. However, the Tg and cAMP levels decreased gradually with the age of the cultures. In the secondary culture the TSH-stimulated Tg production decreased from 253 ng/μg DNA (205-263) after 3 weeks to 18 ng/μg DNA (6-81), P < 0.001, n = 6 after 12 weeks and TSH-stimulated cAMP production from 660 pmol/μg DNA (500-840) to 60 (40-200), P < 0.001, n = 6. The decreased responsiveness of long-term cultures results in preference of short-term secondary cultures, which provide a more suitable experimental model for in vitro investigation of human thyroid cell functions.