Most isolated smooth muscle cells from guinea pig taenia caecum responded repeatedly, showing an all-or-none response to acetylcholine (ACh). However, the average responses of all the cells were graded owing to the difference in the threshold concentration of ACh, like that of whole tissue. The sensitivity of the muscarinic receptor on individual cells was the same as that of whole tissue and ACh bound to the receptor concentration-dependently. The shortening of the cells in response to ACh depended upon the influx of extracellular Ca²⁺ through the Ca channel. ⁴⁵Ca²⁺ influx stimulated by ACh was very sharp and highly correlated with the shortening of the cells. The shortening of α-toxin-permeabilized smooth muscle cells was induced by increasing free Ca²⁺. The concentration-response relationship to free Ca²⁺ had a very steep slope, and the shortening appeared to be an all-or-none response rather than a graded response. In conclusion, it is suggested that isolated smooth muscle cells show an all-or-none response as a result of a slight increase in the intracellular free Ca²⁺ level over the threshold concentration when early signalling coupled to ACh-receptor stimulation reaches the threshold to evoke Ca²⁺ influx and Ca²⁺ release.