1. Properties of the hyperpolarization-activated cation current (Ih) were investigated in thalamocortical neurones of an in vitro slice preparation of the rat ventrobasal thalamic complex (VB) before and during changes of pipette pH (pHp), intracellular pH (pHi) and bath pH (pHb) using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. Recording of Ih with predefined pHp revealed significant shifts in the voltage dependence of Ih activation (V ) of 4-5 mV to more positive values for a pHp of 7.5 and 2-3 mV to more negative values for a pHp of 6.7 as compared to control values (pHp = 7.1). 3. Application of the weak acid lactate (20 mM), which produced a slow monophasic intracellular acidification, induced a reversible negative shift of V of up to 3 mV. Application of 20 mM TMA, which caused a distinct intracellular alkalinization, shifted V to 4-5 mV more positive values. 4. In slices bathed in Hepes-buffered saline, no significant pHo dependence of Ih was observed. Changing pHo by altering the extracellular [HCO3-] in the presence of constant pCO2 also revealed no significant pHo dependence of Ih. 5. Rhythmic stimulation of thalamocortical neurones with repetitive depolarizing pulse trains caused an intracellular acidification, which reversibly decreased the amplitude and time course of activation of Ih. 6. The results of the present study indicate that shifts in pHi result in a significant modulation of the gating properties of Ih channels in TC neurones. Through this mechanism activity-dependent shifts in pHi may contribute to the up- and downregulation of Ih.