Synthesis of ecdysteroid molting hormones by crustacean Y-organs is believed to be regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. In the present study, steady-state MIH mRNA and hemolymph ecdysteroid levels were determined by Northern blot and radioimmunoassay, respectively, during the molt cycle of the blue crab, Callinectes sapidus. The level of MIH mRNA dropped steadily during premolt (D1-D4), reaching a minimum in D3/D4, then increased by 10-fold in postmolt (A/B) and remained elevated during intermolt (C4). These stage-specific changes in MIH mRNA levels were accompanied by significant fluctuations in the hemolymph ecdysteroid titer. The ecdysteroid titer increased steadily to a peak of 377.0 ng/ml in D3 of premolt, then dropped to 120.0 ng/ml in D4 (just prior to molting), and was low during postmolt (A/B, 4.4 ng/ml) and intermolt (C4, 3.3 ng/ml). The results represent the first report of developmental changes in MIH gene expression and are generally consistent with the hypothesis that MIH negatively regulates ecdysteroid synthesis in crustaceans.