At equilibrium (4 h incubation), [3H]TCP (N-(1-[2-thienyl]-cyclohexyl)-3,4-[3H]piperidine) binding to well-washed rat forebrain membranes was enhanced in a concentration-dependent and 2-APV (2-amino-5-phosphonovaleric acid)-sensitive fashion by L-glutamate (EC50 = 0.2 microM; maximal effect +280%). L-glutamate (10 microM) increased the affinity of [3H]TCP from 78 to 28 nM, but was without effect on the maximal binding capacity. The enhancing effect of L-glutamate on [3H]TCP binding was potentiated by glycine in a concentration-dependent manner (EC50 = 50 nM, maximal effect +30% in the presence of 10 microM L-glutamate; EC50 = 2 microM, maximal effect +29% in the presence of 0.1 microM L-glutamate). This effect was strychnine-insensitive. Glycine failed to enhance [3H]TCP binding in the presence of 10 microM 2-APV. The glycine effect was due to an increase in affinity (Kd = 21 nM in the presence of 10 microM glycine and 10 microM L-glutamate); glycine did not affect the maximal binding capacity. The glycine enhancement of L-glutamate-stimulated [3H]TCP binding was not antagonised by 1 microM strychnine and was mimicked by L-serine and L-alanine but not by GABA, taurine or beta-alanine. Kinetic analysis of the glycine and L-glutamate enhancement of [3H]TCP binding indicated that the L-glutamate effect was related to a decrease in the [3H]TCP dissociation rate while the glycine effect was due to an increase in the rate of [3H]TCP association in the presence of L-glutamate.