Preoptic magnocellular neurosecretory cells (NSCs) in the brain of rainbow trout show synchronization of periodic Ca(2+) pulses, patterns of which differ between vasotocin (VT) and isotocin (IT) neurons. To provide neuroanatomical bases of the synchronized periodic Ca(2+) pulses and their biological implications, we examined the organization of preoptic VT and IT neurons in the brain of rainbow trout. The cytoarchitecture of the preoptic neurosecretory system was characterized by a confocal double-color immunofluorescence. Two to five VT neurons, and also IT neurons, aggregate to form cell-type specific clusters. VT clusters tend to localize medially, while IT clusters laterally. VT neurons are closely apposed at the proximal neuronal processes. A Golgi-like immunohistochemistry demonstrated that VT and IT fibers distribute widely in the brain, such as ventral telencephalon, diencephalon, and various mesencephalic structures, in addition to the neurohypophysial projections. Projections from single VT and IT neurons were examined by an intracellular staining with biocytin injection in a sagittally hemisected brain preparation, which contains the entire forebrain region. Single VT and IT neurons project toward the pituitary and the extrahypothalamic regions. Some IT neurons, but not VT neurons, were dye-coupled. These results support the idea that the same types of NSCs are connected to form cell-type-specific networks responsible for the synchronization of periodic Ca(2+) pulses. The organization of the preoptic neurosecretory system shown in the present study is suitable for the simultaneous control of neurohypophysial and extrahypothalamic outputs through the synchronization of electrical activity.