Abstract Chemical, biochemical, and immunohistochemical evidence is reported demonstrating the presence in the brain of the cuttlefish Sepia officinalis of a Ca2+-dependent nitric oxide synthase, NMDAR2/3 receptor subunits, and glutamate, occurring in neurons and fibers functionally related to the inking system. Nitric oxide synthase activity was concentrated for the most part in the cytosolic fraction and was masked by other citrulline-forming enzyme(s). The labile nitric oxide synthase could be partially purified by ammonium sulfate precipitation of tissue extracts, followed by affinity chromatography on 2′, 5′-ADP-agarose and calmodulin-agarose. The resulting activity, immunolabeled at 150 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by antibodies to rat neuronal nitric oxide synthase, depended on NADPH and tetrahydro-L-biopterin, and was inhibited by NG-nitro-L-arginine. NMDAR2/3 subunit-immunoreactive proteins migrating at 170 kDa could also be detected in brain extracts, along with glutamate (whole brain : 0.32 ± 0.03 μmol of glutamate/mg of protein ; optic lobes : 0.22 ± 0.04 ; vertical complex : 0.06 ± 0.06 basal lobes : 0.58 ± 0.04 ; brachial lobe : 0.77 ± 0.06 ; pedal lobe : 1.04 ± 0.08 ; palliovisceral lobe : 0.86 ± 0.05). Incubation of intact brains with 1.5 mM glutamate or NMDA or the nitric oxide donor 2-(N,N-diethylamino)diazenolate-2-oxide caused a fivefold rise in the levels of cyclic GMP, indicating operation of the glutamate-nitric oxide-cyclic GMP signaling pathway. Immunohistochemical mapping of Sepia CNS showed specific localization of nitric oxide synthase-like and NMDAR2/3-like immunoreactivities in the lateroventral palliovisceral lobe, the visceral lobe, and the pallial and visceral nerves, as well as in the sphincters and wall of the ink sac.