Abstract: The effects of some GABA analogues and some drugs on the binding of [3H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron‐enriched culture of embryonic rat brain were examined using Na+‐free Tris‐citrate medium and a centrifugation method. Competition for [3H]muscimol binding sites by excess(10−5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [3H]muscimol binding was potently inhibited by muscimol itself (IC50, 2.5 nM), GABA (1C50, 43 nM), isoguvacine (IC50, 61 nM), and 3‐aminopropanesulphonic acid (IC50, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC50, 800 nM). δ‐ Aminovaleric acid (IC50, 2.6 μM), the glycinelp‐alanine antagonist strychnine (IC50, 6.6 μM), and the predominantly glial GABA uptake inhibitors β‐alanine (IC50, 23 μM) and p‐proline (IC50, 66 μM) also inhibited [3H]muscimol binding. Other inhibitors of Na+‐dependent GABA uptake, (±)‐nipecotic acid, L‐ 2,4‐diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [3H]muscimol binding (IC50≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [3H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.