gamma-Aminobutyric acid (GABA) is a major amino acid neurotransmitter in the vertebrate brain. To provide detailed information on the distribution of the GABA in zebrafish (Danio rerio), neurons were labeled with mCherry driven by the glutamic acid decarboxylase 67 (gad67) promoter. In the transgenic line Tg(gad67: mCherry), mCherry-positive gad67 cell bodies were predominantly localized to the olfactory bulb, pallial zones, subpallium zones, parvocellular preoptic nucleus, periventricular gray zone of optic tectum, torus semicircularis, posterior tuberculum, medial longitudinal fascicle, caudal zone of periventricular hypothalamus, and oculomotor nucleus. mCherry-positive fibers were widely distributed in the olfactory bulbs, subpallium, thalamus, ventral hypothalamic zone, tectumopticum, mesencephalon, and rhombencephalon. mCherry-positive neurons were also observed in the retina and the spinal cord. The anatomical relationships between GABAergic and gonadotrophin-releasing hormone 3 (GnRH3) neurons were investigated by crossing Tg(gad67: mCherry) fish with the previously established Tg(gnrh3: EGFP) transgenic line. GnRH3 cell bodies and fibers were contacted by GABAergic fibers directly in the ventral telencephalon and anterior tuberal nucleus. A subpopulation of GnRH3 neurons in the ventral telencephalic area was also labeled with mCherry, so some GnRH3 neurons are also GABAergic. GABA(B) receptor agonist (baclofen) and antagonist (CGP55845) treatments indicated that GABA(B) receptor signaling inhibited gnrh3 expression in larval fish but was stimulatory in adult fish. The expression of pituitary lh beta and fsh beta was stimulated by intraperitoneal injection of baclofen in adult fish. We conclude that GABA via GABAB receptors regulates GnRH3 neurons in a developmentally dependent manner in zebrafish.