Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 +/- 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.