In crustaceans, molt-inhibiting hormone (MIH) has been proposed to regulate molting by inhibiting the secretion of ecdysteroids from the Y-organ. Thus, MIH titer in the hemolymph should be inversely related to ecdysteroid titers during the molt cycle. However, it has not been demonstrated whether the MIH titer in the hemolymph changes during the molt cycle. The purpose of this study was to determine the changes in the MIH titers in the hemolymph during the molt cycle of the American crayfish, Procambarus clarkii, and to discuss the role of MIH in regulation of molting. As predicted by the hypothesis, the hemolymph MIH titer was high at the intermolt stage when the hemolymph ecdysteroid titer was low, and the MIH titer decreased to a basal level at the early premolt stage when the hemolymph ecdysteroid titer began to increase slightly. At the middle premolt stage when the hemolymph ecdysteroid titer increased, the MIH titer was restored to a level as high as that during the intermolt stage. This is in contradiction to the hypothesis. However, the Y-organs at this stage scarcely responded to MIH both in vitro and in vivo. The present findings suggest that ecdysteroid secretion from the Y-organ may be regulated not only by changes in the hemolymph MIH titer, but also by changes in the responsiveness of the Y-organ to MIH. (C) 2003 Elsevier Inc. All rights reserved.