The possible regulation of guinea pig enterocyte ion channels by GTP-binding proteins has been investigated by using the whole-cell recording mode of the patch-clamp technique and non-hydrolysable analogues of GTP. The main K+ currents in these cells are mediated by inwardly-rectifying K+ channels. Intracellular dialysis with hydrolysis-resistant GTP analogues leads to the deactivation of the inward K+ currents. Non-hydrolysable analogues of ATP or GDP were without effect. The effect occurs after a lag phase of 2 to 7 min, suggesting a multistep mechanism. Cl currents were not affected by any of the nucleotide analogues. It is suggested that inwardly-rectifying K+ currents are deactivated by a G-protein-dependent process.