The mechanism of the effect of intracellular ATP on the hyperpolarization-activated non-selective cation current (I_h) in rat dorsal rootganglion neurons was investigated using a whole-cell voltage-clamp technique. Under voltage-clamp conditions, I_h was activated by hyperpolarizing pulses raised to a voltage of between -70 and -130 mV. The activation curve of I_h in rat dorsal root ganglion (DRG) neurons shifted by about 15 mV in the positive direction with an intracellular solution containing 1 mM cAMP. When ATP (2 mM) was applied intracellularly, the half-maximal activation voltage (V_half) of I_h shifted from -97.4 ± 1.9 mV to -86.8 ± 1.6 mV, resulting in an increase in the current amplitude of I_h by the pulse to between -80 and -90 mV. In the presence of an adenylate cyclase inhibitor, SQ-22536 (100 µM), the intracellular dialysis of ATP also produced a shift in the voltage-dependence of I_h in rat DRG neurons, indicating that the effect of ATP was not caused by cAMP converted by adenylate cyclase. Intracellular dialysis of a non-hydrolysable ATP analog, AMP-PNP or ATP--S, also produced a positive shift in the voltage-dependence of I_h activation, suggesting that the effect of ATP results from its direct action on the channel protein. These results indicate that cytosolic ATP directly regulates the voltage dependence of I_h activation as an intracellular modulating factor.