1. Cationic current (Icat) was evoked in single isolated smooth muscle cells either by activating muscarinic receptors with the stable muscarinic agonist, carbachol (CCh), or by dialysing cells with GTP-gamma S. It was studied using patch-clamp recording techniques in cells obtained by enzymatic digestion from the longitudinal muscle layer of the guinea-pig small intestine. 2. Icat appears only when muscarinic receptors or G-proteins are activated, but it is strongly voltage-dependent. Its activation could be described by the Boltzmann equation. During desensitization of Icat evoked by 50 microM CCh, the slope factor, k, remained constant whereas the maximal conductance, Gmax, slowly decreased and the potential of half-maximal activation, V1/2, shifted positively by 32 mV during 4 min. 3. At peak response either to extracellular application of CCh (GTP-free, or 1 mM GTP-containing, pipette solution) or to intracellular application of GTP-gamma S (no CCh), the size and voltage-dependent properties of Icat were similar. However, Icat desensitization was slower in the presence of GTP (CCh applied) in the pipette solution and much slower with GTP-gamma S in the pipette (no CCh) compared to GTP-free pipette solution (CCh applied); the decrease in Gmax with time was much delayed and the positive shift of the activation curve was inhibited. GDP-beta S added to the pipette solution at 2 mM abolished Icat in response to applied CCh; 50 microM did not prevent Icat generation but significantly accelerated desensitization. 4. It was concluded that the rate of desensitization of the carbachol-evoked cationic current was due to a decline in the concentration of activated G-protein in the cell, which reduced the maximum number of channels which could be opened and shifted their activation range to less negative potentials.