The distribution of γ-aminobutyric acid (GABA)-containing neurons and nerve fibers was studied in the isthmic nucleus of the frog Rana esculenta using light and electron microscopical immunohistochemical techniques. Approximately 0.5% of isthmic cells showed GABA immunopositivity, and the majority of these cells was found in the anterior one-third of the nucleus. A meshwork of GABA-immunostained fine beaded axons filled the entire isthmic nucleus. The GABA-immunoreactive terminals formed pericellular basket-like structures around a few cells both in the medulla and the cortex of the isthmic nucleus. To determine the source of GABA-positive fibers in the isthmic nucleus lesion experiments were carried out. After unilateral tectal ablation no change was observed in GABA immunoreactivity. Hemisectioning the tegmentum close to the anterior border of the isthmic nucleus, transection of the caudal tectal commissure and decussatio veli, or electrical lesioning of the anterodorsal tegmental nucleus all resulted in a moderate decrease in the density of GABA-positive fibers. Our results suggest that the majority of GABA-positive fibers derives from local GABA-positive cells, but some GABAergic afferents seem to arise in the tegmentum.