Previous studies have shown that thyroid cells in monolayer culture exhibit bidirectional ion transport comprising apical-to-basal amiloride-sensitive Na+ transport and oppositely directed bumetanide-sensitive Cl- transport. We have now investigated the role of ion transport in the regulation of thyroid follicular size using follicular primary porcine thyroid cell cultures. Bumetanide (10 microM) added at the beginning of culture inhibited the formation of follicular lumina and caused a fall in follicle height when added to 3-day-old cultures. In contrast, phenamil (1 microM; an amiloride analog) increased follicle size both in freshly isolated and 3-day-old cultures. The effect of bumetanide was prevented by the prior addition of phenamil. Micropuncture studies showed that follicles had a lumen-negative, basal-positive transepithelial potential difference which was progressively reduced in magnitude by the serial addition of bumetanide (10 microM) and phenamil (1 microM). We conclude that thyroid follicles possess a bidirectional ion transport system which transports Na+ in an apical-to-basal direction and Cl- in the opposite direction. The balance between these two processes determines net solute flux and hence follicular size. A physiological role of ion transport in the thyroid may be to regulate follicular volume suggesting that abnormalities of ion transport may be responsible for disorders of follicular size.