Myenteric neurones from 1-10-day-old rats were isolated from the small and large intestine by enzymatic digestion with collagenase. Single cells were collected and kept in culture for up to 1 week. After 1-5 days in culture, membrane potential and ionic currents were measured with the whole-cell patch-clamp technique. The intracellular Ca2+ concentration was measured with the fura-2 method. The short-chain fatty acid butyrate (50 mmol L-1) induced a reversible hyperpolarization of the myenteric neurones by about 10 mV. This hyperpolarization was concomitant with an inhibition of a TTX-sensitive Na+ current. The hyperpolarization could be suppressed by intracellular application of Cs+, a nonselective K+ channel blocker. Fura-2 experiments revealed that butyrate induced an increase of the intracellular Ca2+ concentration. The butyrate response was suppressed by thapsigargin, indicating that butyrate stimulates the release of intracellular Ca2+. This release is responsible for the voltage response, because intracellular chelation of Ca2+ inhibited the butyrate induced hyperpolarization. Consequently, butyrate acts on enteric neurones by releasing Ca2+ from intracellular stores with the consequence of the activation of K+ channels, followed by a hyperpolarization.