We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide is present in the nerve ganglion of the chiton Acanthopleura japonica (Mollusca, Polyplacophora) using reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoas-say (TR-FIA) analysis, and immunohistochemistry. An extract of the chiton head region showed a similar retention time to that of synthetic lamprey GnRH-II on rpHPLC combined with TR-FIA analysis using a rabbit polyclonal antibody raised against chicken GnRH-II (aCII6). Cell bodies immunostained with LRH13 (a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the cerebrobuccal ring (CBR). Cell bodies immunostained with aCII6 were not only observed in the CBR but also in the lateral nerve cord (LCo). Fibers immunostained with LRH13 and aCII6 were widely distributed throughout the central nervous system in the CBR, subradular ganglion (SubRG), pedal nerve cord (PCo), pedal commissure (P/PCom), lateropedal commissure (L/PCom), and from the LCo to the suprarectal commissure (SupRecCom). The cell bodies and fibers immunostained with these two antisera were distinguishable by dual-label immunohistochemistry. These results suggest that multiple GnRH-like peptides are present in the nerve ganglion of the chiton Acanthopleura japonica.