Our previous report demonstrated that chick myoblasts are equipped with Ca2⁢-permeable stretch- activated channels and Ca2⁢-activated potassium channels (KCa), and that hyperpolarization-induced by KCa channels provides driving force for Ca2⁢ influx through the stretch-activated channels into the cells. Here, we showed that acetylcholine (ACh) also hyperpolarized the membrane of cultured chick myoblasts, suggesting that nicotinic acetylcholine receptor (nAChR) may be another pathway for Ca2⁢ influx. Under cell-attatched patch configuration, ACh increased the open probability of KCa channels from 0.007 to 0.055 only when extracellular Ca2⁢ was present. Nicotine, a nAChR agonist, increased the open probability of KCa channels from 0.008 to 0.023, whereas muscarine failed to do so. Since the activity of KCa channel is sensitive to intracellular Ca2⁢ level, nAChR seems to be capable of inducing Ca2⁢ influx. Using the Ca2⁢ imaging analysis, we were able to provide direct evidence that ACh induced Ca2⁢ influx from extracellular solution, which was dramatically increased by valinomycin-mediated hyperpolarization. In addition, ACh hyperpolarized the membrane potential from ⁣12.5⁑3 to ⁣31.2⁑5 mV by generating the outward current through KCa channels. These results suggest that activation of nAChR increases Ca2⁢ influx, which activates KCa channels, thereby hyperpolarizing the membrane potential in chick myoblasts.