The insect brain offers a unique opportunity for cell biological investigation of calcium signalling in relatively homogeneous neuronal populations, such as the mushroom body neurons. This review summarizes fluorescence imaging experiments with the indicator dye fluo-3 to investigate the calcium responses to transmitter stimulation of honeybee mushroom body neurons in primary culture. Application of acetylcholine (ACh) or nicotine promoted a calcium influx into the cell body and neurites. The increase in intracellular calcium after ACh stimulation was blocked by alpha-bungarotoxin. These results support previous histochemical studies that suggested the expression of nicotinic cholinergic receptors on Kenyon cells. An increase in cytoplasmic calcium levels leads in specific neurons to the generation of nitric oxide (NO) by a Ca2+/calmodulin activated NO synthase. NO is thought to diffuse as a short-lived messenger molecule through the plasma membrane. Using a sensitive photometric assay it could be shown that dissociated cells from the locust brain release NO after stimulation by agents elevating cytoplasmic Ca2+ levels and by the excitatory neurotransmitter ACh. The experiments in dissociated culture therefore allow the demonstration of a Ca(2+)-dependent release during nerve cell depolarization, which is a basic requirement for identifying NO as a messenger molecule.