Water and electrolyte contents, cell pH, membrane potential and 125I- uptake were determined in cultured follicular cells of turtle thyroid. The Na+, K+ and Cl- concentrations in the cultured thyroid cells were 59.2, 119.0 and 50.9 mmol/l cell water respectively. Treatment with TSH (10 mu./ml for 24 h) increased the K+ and Cl- and decreased the Na+ concentrations in cells. The water and protein contents of these cells were 81.6 and 8.7 g/100 g cells respectively. The cell pH was 6.91. With glass micro-electrodes, the resting membrane potential of thyroid cells cultured in Medium 199 averaged 33.9 +/- 0.63 mV which is slightly higher than 29.8 +/- 1.6 mV as calculated from the data on the uptakes of [14C]methyltriphenylphosphonium and 3H2O by the cells. The potential varied linearly with the log of external K+ concentration (between 15 and 120 mmol/l) with a slope of about 24 mV per tenfold change in K+ concentration. Both TSH and cyclic AMP depolarized the cell membrane. Calculations based on the values for the electrolyte concentrations in cells and in culture medium indicated that Na+, K+ and Cl- were not distributed according to their electrochemical gradients across the cell membrane. Na+ was actively transported out of the cells and K+ and Cl- into the cells. Follicular cells of turtle thyroid cultured in the medium without addition of TSH formed a monolayer. Their iodide-concentrating ability was low and they did not respond to TSH with an increase in iodide uptake. In contrast, cells cultured in medium containing TSH tended to aggregate and organize to form follicles. They had higher ability to concentrate iodide and respond to TSH.