Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca2+ in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca2+ in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca2+ was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5 ng/mL on D0 to 49.6 ng/mL on D6 and 87.2 ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca2+. J. Exp. Zool. 315:56–60, 2011. © 2010 Wiley-Liss, Inc.