Differentiation and morphogenesis of skeletal muscle are complex and asynchronous events that involve various myogenic cell populations and extracellular signals. Embryonic and fetal skeletal myoblasts are responsible for the formation of primary and secondary fibers, respectively, although the mechanism that diversifies their fate is not fully understood. Calcium transients appear to be a signaling mechanism that is widely utilized in differentiation and embryogenesis. In mature skeletal muscle, calcium transients are generated mainly by ryanodine receptors (type 1 and type 3),which are involved in excitation-contraction coupling. However, it is not clear whether the activity of these receptors is important for contractile activity alone or whether it may also play a role in regulating the differentiation/developmental processes. To clarify this point, we first examined the expression of the receptors during development. The results show that the expression of both receptors appears as early as E13 during limb muscle development and parallels the expression of skeletal myosin. The expression and the activity of both receptors is maintained in vitro by all myogenic cell populations isolated from different stages of development,including somitic, embryonic and fetal myoblasts and satellite cells. Blocking ryanodine receptor activity by using ryanodine inhibits in vitro differentiation of fetal myoblasts (judged by the expression of sarcomeric myosin and formation of multinucleated myotubes) but not of somitic or embryonic and satellite muscle cells. This block is caused by the transcriptional inhibition of markers characteristic of terminal differentiation, rather than commitment, as the expression of muscle regulatory factors is not impaired by ryanodine treatment. Taken together, the data reported in this paper demonstrate that, although calcium transients represent a general mechanism for the control of differentiation and development, multiple calcium-dependent pathways may be relevant in different myogenic populations during development. Moreover, since fetal myoblasts are responsible for the formation of secondary fibers during development, and therefore for the building of the bulk of muscular mass, these results suggest that calcium release from ryanodine receptors plays a role in the histogenesis of mammalian skeletal muscle.