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1. The effects of Na-free solutions (using Li, Tris, sucrose or Mg as Na substitutes) on the contractile responses, membrane depolarization and 42K efflux produced by carbachol in the smooth muscle of the guinea-pig taenia coli have been investigated. The effect of these Na-free solutions on intracellular ion content of the muscle has also been studied. 2. Na removal induced a pattern of changes in the tone of the muscle characteristic of the substitute used, probably reflecting changes in transmembrane Ca fluxes involving Na. 3. Contractile responses to 10 sec application of 5 x 10(-5) M-carbachol were greatly reduced in Na-free solutions with all the Na substitutes used. This did not correlate with reduction in membrane depolarization or 42K efflux produced by the drug in the various Na-free medial used. 4. Intracellular Na seems important for maintaining the contractile response, since in Na-free solutions cellular Na levels and contractile responses were better maintained at 13 degrees than 34 degrees C and in tissues stimulated with carbachol every 10 min the final magnitude of the response was related to cellular Na content. If, however, the tissues was left unstimulated in Na-free Mg or sucrose solution a large response could still be obtained when cellular Na content was very low. A model is described which could account for these results in terms of an intracellular Ca store released by carbachol and requiring intracellular Na. 5. In tissues continuously exposed to 10(-4) M-carbachol Na removal, even for only 10 sec, produced rapid relaxation, probably secondary to changes in electrical properties of the membrane caused by removal of external Na.