In order to determine the titer of molt-inhibiting hormone (Prc-MIH) in the hemolymph of the American crayfish Procambarus clarkii, a time-resolved fluoroimmunoassay (TR-FIA) was established using specific antibodies against N-terminal and C-terminal segments of Prc-MIH. The lowest limit of detection of Prc-MIH in TR-FIA was 10 amol/assay. The Prc-MIH titers in the hemolymph were 6.53 fmol/ml at the intermolt stage and 1.25 fmol/ml at the early premolt stage. This result is consistent with the long-known hypothesis that the Y-organ is inhibited by MIH during the intermolt stage, whereas the Y-organ is activated by being freed from the inhibitory regulation of MIH.