Intracellular recordings of membrane potential were made from the longitudinal muscle of guinea-pig terminal ileum. It was observed that ouabain or potassium-free solution depolarized the membrane. Upon readmitting potassium to potassium-free solution, the membrane potential rapidly increased. This response was blocked by ouabain and was potentiated in chloride-deficient solution, suggesting that it was due to the activity of an electrogenic sodium pump. When acetylcholine was applied, and then washed from the tissue, there followed a period of increased negativity of the membrane potential, an after-hyperpolarization. This did not occur when responses to acetylcholine were obtained in the presence of ouabain, in potassium-free solution, or in sodium-deficient solution, but the after-hyperpolarization was increased in size in chloride-deficient solution. During a 2 min application of carbachol, the membrane potential fell more rapidly in the presence of ouabain (10 -5 mol/1); this could be explained if sodium pump activity is important in retarding the decline of the sodium and potassium gradients that occurs at this time. It was concluded that the application of acetylcholine or carbachol to ileal muscle increases internal sodium and probably external potassium concentrations. These increases stimulate the activity of the electrogenic sodium pump so that, when the membrane resistance recovers, there is an increased electrogenic contribution to the membrane potential. This produces the after-hyperpolarization which is a feature of the response to acetylcholine.