Abstract We report here the neuron-specific co-expression of four genes coding for neuropeptides involved in the control of male behaviour. These neurons are located in the anterior lobe of the right cerebral ganglion in the central nervous system of Lymnaea stagnalis and project via the penis nerve to the penial complex. In order to accomplish optimal assurance we applied in situ hybridization, immunocytochemistry and matrix-assisted laser desorption ionization mass spectrometry. The anterior lobe neurons express the gene encoding the amidated tetrapeptide APGWamide. Subsets of these cells are now shown to co-express the APGWamide gene exclusively with one of three other neuropeptide genes, encoding Lymnaea neuropeptide Y, conopressin or pedal peptide, respectively. All four genes are also expressed in other neurons in other centres projecting to the penial complex, but in these cells co-expression was not observed. The neuropeptides encoded by the genes could be identified in the anterior lobe cell bodies on the basis of immunocytochemistry and mass spectrometrical analysis. The neuropeptides APGWamide and Lymnaea neuropeptide Y, which are co-localized in the anterior lobe cells as well as in axons innervating the penis retractor muscle, do not induce muscle contraction but have a modulatory action by affecting the relaxation rate and amplitude of the contraction. APGWamide and conopressin had earlier been suggested to modulate peristalsis of the vas deferens. Thus, it seems that the neurons co-expressing the various combinations of neuropeptide genes in the anterior lobe represent functional units, each acting in the fine tuning of different muscles involved in specific aspects of male copulation behaviour.