Neuropeptides are widely distributed throughout the nervous system and exert a large number of heterogeneous functions. While they are synthesized in the soma, release is thought to take place in axonal terminals of neurons. A good model system to investigate the role of peptides in the nervous system is provided by the central posterior/prepacemaker nucleus (CP/PPn) of pacemaker nucleus (Pn), a medullary cell group controlling the electric organ discharge (EOD). Previous immunocytochemical and in situ-hybridization studies employing topographical criteria indicated that PPn neurons may express the neuropeptide somatostatin (SS). In the present study, we unambiguously identified PPn neurons by in vitro tract tracing. By combining this technique with SS immunocytochemistry, we found that a large portion of retrogradely labelled PPn neurons exhibited SS-like immunoreactivity (72-89%, n = 708 cells in 10 fish examined). Surprisingly, however, neither the proximal PPn axons nor anterogradely labelled terminals innervating the Pn displayed significant amounts of SS-like immunolabelling (n = 10 fish examined in each experiment). These results and the lack of SS binding sites in the Pn [82] suggest that SS expressed by PPn cells is not synaptically released at the target site of their axons, the Pn, but acts via a nonsynaptic mechanism in the CP/PPn proper.