Nitric oxide (NO), hydrogen sulfide (H₂S) and carbon monoxide (CO) are thought to act as gaseous neurotransmitters or neuromodulators in the brain across species.  Nitric oxide (NO) acts as a neuromodulator in higher-order brain functions such as learning and memory; and H₂S and CO play as neuromodulators in induction of long-term potentiation.  In the brain of honeybee (Apis mellifera), NO-mediated signaling pathway is involved in olfactory discrimination and memory formation.   However, gaseous neurotransmitter/modulator-related genes have not yet been characterized from the honeybee brain.    In the present study, we cloned full-length cDNAs encoding nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), cystathionine beta-synthase (CBS) and heme oxygenase (HO) from the honeybee brain.  We also investigated the distribution of NOS mRNA and NOS protein in the honeybee brain by in situ hybridization and NADPH diaphorase histochemistry. The honeybee brain contains at least one gene for each of NOS, CBS and HO.  The deduced proteins for NOS, CBS and HO are thought to contain domains to generate NO, H₂S and CO, respectively.  The honeybee brain contains three subunits of sGC: sGCa1, sGCb1 and sGCb3.  Phylogenetic analysis of sGC revealed that Apis sGCa1 and sGCb1 are closely related to NO- and CO-sensitive sGC subunits, whereas Apis sGCb3 is closely related to insect O₂-sensitive sGC subunits.  In addition, NOS mRNA was exclusively localized in the mushroom bodies and the optic lobes.  NOS activity was detected in the antennal lobes, the optic lobes, the mushroom bodies, the central body complex and the lateral protocerebral lobes.  Our results suggest that NO-mediated signaling pathway is involved in various brain functions and H₂S and CO can be endogenously produced in the honeybee brain.