The temperature dependence of hyperpolarization-activated current (I_h) was investigated in freshly isolated guinea pig ileal smooth muscle cells, using the nystatin-perforated whole cell recording technique. Hyperpolarizing pulses (50 to 120 mV) from 40 mV evoked time-dependent inward rectifying currents with a reversal potential of 33 mV and a slow activation time course well approximated by a single exponential. The properties of these currents, such as steady-state variables, dependence on external K, modification by norepinephrine, and blockade by Cs or ZD-7288, coincide well with those of the "classical" I_h discovered in the sinoatrial node. Raising the temperature (range: 22-33°C) accelerated the activation time course of this I_h and shifted its 50% activation potential positively (12 mV/10 degree) with much less change in the maximum conductance. Based on a simple closed-open model, this can be explained by a high temperature dependence of the opening rate constant (temperature coefficient: 3.4). The activation profile of reconstructed I_h at 36°C suggests that a considerable overlap could occur between the ranges of I_h activation and physiological membrane potential.