BACKGROUND: Butterfly wing color patterns emerge as the result of a regular arrangement of scales produced by epithelial scale cells at the pupal stage. These color patterns and scale arrangements are coordinated throughout the wing. However, the mechanism by which the development of scale cells is controlled across the entire wing remains elusive. In the present study, we used pupal wings of the blue pansy butterfly, Junonia orithya, which has distinct eyespots, to examine the possible involvement of Ca(2+) waves in wing development. RESULTS: Here, we demonstrate that the developing pupal wing tissue of the blue pansy butterfly displayed spontaneous low-frequency Ca(2+) waves in vivo that propagated slowly over long distances. Some waves appeared to be released from the immediate peripheries of the prospective eyespot and discal spot, though it was often difficult to identify the specific origins of these waves. Physical damage, which is known to induce ectopic eyespots, led to the radiation of Ca(2+) waves from the immediate periphery of the damaged site. Thapsigargin, which is a specific inhibitor of Ca(2+)-ATPases in the endoplasmic reticulum, induced an acute increase in cytoplasmic Ca(2+) levels and halted the spontaneous Ca(2+) waves. Additionally, thapsigargin-treated wings showed incomplete scale development as well as other scale and color pattern abnormalities. CONCLUSIONS: We identified a novel form of Ca(2+) waves, spontaneous low-frequency slow waves, which travel over exceptionally long distances. Our results suggest that spontaneous Ca(2+) waves play a critical role in the coordinated development of scale arrangements and possibly in color pattern formation in butterflies.