NADPH diaphorase (NADPHd) is a marker enzyme for nitric oxide (NO)-producing cells in vertebrates. This paper investigates the relationship between NADPHd and the NO-producing enzyme NO synthase (NOS) in neuronal tissue of Apis and Drosophila, two insects used for studying learning. First, the NOS and the NADPHd in both species were characterized biochemically. The fixation-insensitive NADPHd activity, which accounts for the staining in NADPHd histochemistry, co-purifies with the insect Ca2+/calmodulin-dependent NOS. Formation of NO from L-arginine depends on NADPH, and half-maximal stimulation is observed with 0.3 microM Ca2+. NOS is competitively inhibited by methyl-L-arginine and nitro-L-arginine, with Ki of 1.7 and 1.9 microM, respectively. The co-purification and the competitive inhibition of NOS by the NADPHd substrate, nitro blue tetrazolium (NBT), are proof that in insects the enzyme responsible for fixation-insensitive NADPHd activity is nitric oxide synthase. Second, the NOS activity was quantified in distinct neuropiles and the NO-producing neuropiles were visualized using NADPHd histochemistry. In both species the highest NOS activity is found in the chemosensory neuropiles of the antennal lobes, intermediate activity in the neuropiles of the central brain and by far the lowest NOS activity in the visual neuropiles. Although in both species the Kenyon cell somata of the mushroom bodies show no detectable staining, the neuropiles of the mushroom bodies of Drosophila and Apis show a distinct staining. The staining pattern of NOS in both species is different to that of all known neurotransmitters.