It has become a general notion that there are multiple GnRH systems in the vertebrate brains. To measure GnRH release activities from different GnRH systems, we conducted a static incubation of brain-pituitary slices under various conditions, and GnRH released into the incubation medium was measured by RIA (RIA). The slices were divided into two parts, one containing GnRH neurons in the preoptic area and axon terminals in the pituitary (POA-GnRH slices), and the other containing the cell bodies and fibers of terminal nerve-GnRH neurons and midbrain tegmentum-GnRH neurons (TN-TEG-GnRH slices). We demonstrated that GnRH release was evoked by high [K⁺]_o depolarizing stimuli (in both slices) via Ca²⁺ influx through voltage-gated Ca²⁺ channels. The most prominent result was the presence of conspicuous sexual difference in the amount of GnRH release in the POA-GnRH slices. The GnRH release from TN-TEG-GnRH slices also showed a small sexual difference, which was by far inconspicuous than that of POA-GnRH slices. Immunohistochemical analysis using an antiserum specific to the seabream GnRH (sbGnRH; suggested to be specific to POA-GnRH neurons) revealed presence of much larger number of POA-GnRH neurons in males than in females. This clear morphological sexual difference is suggested to underlie that of GnRH release in the POA-GnRH slices.