The mechanism of intracellular calcium ions (Ca(2+)) increase in chicken myoblasts was studied using histological, immunohistochemical, immunoblotting and Ca(2+) imaging techniques. Mononuclear myoblasts at embryonic day 12 (E12) contained myofibrils in the peripheral cytoplasm, and the sarcoplasmic reticulum was observed in the cytoplasm. Several Ca(2+)-related receptors, namely acetylcholine (ACh) receptors, dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), were detected in the tissue as early as E12. Western blotting analyses detected one band corresponding to RyR subtype 3 (RyR3) at E12 and two bands corresponding to RyR1 and RyR3 after E13. Ca(2+) imaging of mononuclear myoblasts in vitro revealed an intense Ca(2+)-increase response to ACh stimulation, and this effect was abolished after EGTA addition to the culture medium. Nifedipine treatment also led to a lack of Ca(2+) increase in response to ACh stimulation, while ryanodine treatment led to a weak Ca(2+)-increase response. On the other hand, multinuclear myoblasts showed a Ca(2+)-increase response to ACh stimulation in the presence of not only EGTA but also nifedipine, although ryanodine treatment led to a lack of Ca(2+) increase. These results suggest that the mechanism of Ca(2+) increase in mononuclear myoblasts involves extracellular Ca(2+) entry through DHPRs, which is amplified by Ca(2+) release from the cytoplasmic Ca(2+) store, while multinuclear myoblasts mainly depend on Ca(2+) release from the cytoplasmic Ca(2+) store.