Motor nerve branches were stimulated in the dermis layer prepared from isolated pieces of dorsal mantle skin of the squid Lolliguncula brevis and the contractions of chromatophore muscle fibers were recorded with the aid of a photo-electric transducer. L-Glutamate (L-Glu), kainate and quisqualate caused a contracture and often repetitive twitch-like contractions. These effects were readily reversible. In the case of L-Glu application, twitches induced by single stimuli applied to motor nerves were enhanced and prolonged. The glutamate antagonists glutamic acid gamma-methyl ester, glutamic acid diethyl ester, D,L-2-amino-4-phosphonobutyrate and gamma-D-glutamylglycine prevented both nerve induced and L-Glu induced contractions. The NMDA-receptor agonists N-methyl-D-aspartate, L-aspartate and D-glutamate, and their antagonists alpha-aminoadipate and D,L-2-amino-5-phosphonovalerate were found ineffective. With the aid of saline media of different Ca and Mg content, it was possible to selectively eliminate one or all components of the effect of L-Glu. Tetrodotoxin abolished nerve induced contractile responses but did not interfere with the contracture caused by L-Glu. Intracellular electrical recording indicated that nerve stimulation causes EPSPs which do not give rise to spike discharges. The results are compatible with the hypothesis that L-Glu is a transmitter substance of the motoneurons that innervate chromatophore muscle fibers.