Spontaneous transient elevations of intracellular calcium (Ca2+i) have functional roles in the development of Xenopus spinal neurons. However, less is known about the roles of elevations of Ca2+i in the differentiation of other cell types. We have examined Xenopus myocytes as a first step in determining if Ca2+i transients are a more general feature of differentiation in excitable cells. We find that cultured myocytes, like neurons, exhibit spontaneous Ca2+i transients during an early developmental period. These transients average 1.4 min in duration and occur at an average frequency of 6/hr in cultures containing myocytes and neurons. Culture conditions can influence transient production; for example, myocyte-enriched cultures have a lower incidence of transient-producing cells. Transients persist in 0-Ca2+ medium, indicating that they arise from intracellular stores. Caffeine-sensitive Ca2+ stores are present in these cells, and depletion or block of these stores eliminates transient production. To determine if transients play a functional role during development, we blocked their production with intracellular BAPTA, a rapid Ca2+ chelator. Cellular differentiation is significantly inhibited only when BAPTA is applied early in development, during the period of transient production, while later BAPTA treatments have no effect. Blocking transient production severely perturbed myofibril organization and sarcomere assembly. However, other aspects of myocyte differentiation were not affected by transient blockade, indicating that not all myogenic differentiation programs are regulated in this manner. Our results suggest that spontaneous Cai2+ transients play a role in cytoskeletal organization during myofibrillogenesis.