[3H]1-Aminocyclopropanecarboxylic acid (ACPC) exhibits high affinity, specific binding to strychnine-insensitive glycine receptors. In extensively washed rat forebrain membranes, the specific binding of [3H]ACPC was optimal at 25 degrees C in the presence of 10 mM MgCl2. Comparable levels of specific [3H]ACPC binding were obtained using centrifugation and filtration for separation of bound from free radioligand. [3H]ACPC labels two sites with Kdl and Bmax1 values of 129 +/- 34 nM and 2.30 +/- 0.37 pmol/mg protein and Kd2 and Bmax2 values of 7.26 +/- 1.69 microM and 20.6 +/- 2.2 pmol/mg protein for the high and low affinity sites, respectively. The Kd of [3H]ACPC (66 nM) estimated under non-equilibrium conditions (koff = 8.91 +/- 0.78 x 10(-3) s-1; kon = 1.35 x 10(-4) nM-1 s-1) was similar to the value obtained for the high affinity site obtained by equilibrium binding. The Kd1 of[3H]ACPC is in good agreement with the previously reported Ki values of ACPC to inhibit the binding of other glycinergic ligands including [3H]glycine, [3H]5,7-dichlorokynurenic acid (5,7-DCKA) and [3H]L-689,560 ((+/-)-4-(trans)-2-carboxy-5,7-dichloro-4- phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline). Moreover, the potencies of a series of glycine site ligands, including glycine. ACPC, 1-aminocyclobutanecarboxylic acid (ACBC), 5,7-DCKA, 7-chlorokynurenic acid (7-CKA), R(+)-3-amino-1-hydroxy-2- pyrrolidine (HA-966) and D-serine, to inhibit [3H]ACPC binding were highly correlated with their potencies to inhibit [3H]glycine and [3H]5,7-DCKA binding (r2 = 0.98-0.51). These results demonstrate that [3H]ACPC is a useful tool for examining the neurochemical and pharmacological properties of strychnine-insensitive glycine receptors.