In mouse C2C12 myotubes acetylcholine (ACh) elevates the concentration of myoplasmic Ca2+ ([Ca2+]i) by inducing Ca2+ influx through transmitter-gated and voltage-gated channels, and by mobilizing Ca2+ from internal stores. The relative contribution of each of these ACh-activated sources to the global [Ca2+]i elevation was estimated. We found that Ca2+ entry through voltage- and ACh-gated channels accounts for roughly 80% of the total [Ca2+]i increment, while mobilization from internal caffeine-sensitive and inositoltrisphosphate- (InsP3-) sensitive stores contributes the remaining 20% to the maximal [Ca2+]i increment. Furthermore, we found that ACh-induced mobilization from InsP3-sensitive stores also develops in embryonic chick myotubes. The differential importance of the Ca2+ signalling pathways activated by ACh during myogenesis is discussed.