Previous autoradiographic studies aimed at showing neurones using GABA as their neurotransmitter have been hampered by the fact that the substance is a ubiquitous metabolite and therefore accumulated by a large variety of cells, including glia. Consequently, GABA uptake markers without this widespread uptake are desirable, and one, [(3)H]isoguvacine, has shown promising results in autoradiographic experiments. Its uptake has now been further studied with quantitative radiochemical techniques. The uptake of the drug was slow compared to GABA uptake and reached a tissue/medium ratio of about 3 after 120 min. The uptake could be inhibited by GABA, beta-alanine or ouabain, and by incubating the retinas at 0 degrees C. The uptake kinetics were complex but suggested a high affinity uptake system (K(m) about 10(?8) M) and perhaps one or several systems with lower affinities. The results indicate that [(3)H]isoguvacine and [(3)H]GABA are accumulated and retained by the same neurones, which most likely use GABA as their neurotransmitter.