The membrane potentials of bone cells derived from calvaria of new born rats was shown to be strongly dependent on temperature. When we lowered the temperature from 36Β°C to 26"C, cells with spontaneous resting membrane potentials (MP) of -80 to -50 mV depolarized (mean amplitude 8 mV; n = 33), and the membrane resistance increased by -80% (n = 20). The temperature response depended on the actual MP, the reversal potential being in the range of -80 to -90 mV. With the application of ouabain (0.1-1 mmoliliter; n = 121, cells depolarized. Simultaneously, the reversal potential of the temperature response was shifted towards more positive values and approached the actual MP level of the cells. Consequently, the depolarization amplitudes induced by lowering temperature were reduced at spontaneous MP levels. The rise of the membrane resistance during cooling was unaffected. When the extracellular chloride concentration was reduced from 133 to 9 mmoliliter, temperature-dependent depolarizations persisted at spontaneous MP values (n = 5). The findings indicate that the marked effects of temperature changes on the MP of bone-derived cells dre mainly determined by changes of the potassium conductance.