Resting intracellular calcium levels and intracellular calcium transients induced by three types of stimulus (acetylcholine, high potassium and caffeine) were recorded, during in vitro myogenesis, by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. Resting levels seemed to decrease with the age of cultured cells and the depolarization-induced transients, through 100 mM K+ or Ach application, were progressively faster and larger as the muscle cells developed. An additive mechanism, likely due to calcium entry into the cell through nicotinic acetylcholine receptors, could explain the differences observed in Ach-induced responses as compared with the 100 mM K(+)-induced ones. In myoballs (the older cells) the calcium transients exhibited progressively a biphasic shape. From data obtained in different conditions (tetrodotoxin, nifedipine, strontium and free Ca EGTA) and those indicating the appearance of caffeine-releasable intracellular calcium stores only at 2-3 days stage, and from the previously reported developmental appearance of calcium currents and contraction, it was proposed that, in young myotubes, the calcium transients were more dependent on extracellular calcium than in older cells. These developmental data are discussed in the light of a known model of the in situ biogenesis of the structures involved in excitation-contraction coupling (ECC) like transverse tubules and triads.