Racemic (dl)-methadone has antagonist activity at the N-methyl-D-aspartate (NMDA) receptor. We evaluated dl-methadone, the opioid active (l-) and the opioid inactive (d-) isomers in competition binding assays. dl-Methadone and its d- and l- isomers exhibited low micromolar affinities for the [3H]MK-801-labeled non-competitive site of the NMDA receptor in both rat forebrain and spinal cord synaptic membranes, with Ki values and displacement curves similar to those of dextromethorphan, an established NMDA receptor antagonist. They lacked affinity at the [3H]CGS-19755-labeled competitive site of the NMDA receptor. Therefore, both methadone and its the d- and l- isomers differ from morphine, hydromorphone, and naltrexone in that they have non-competitive antagonist activity at the NMDA receptor. A non-opioid NMDA receptor antagonist, such as d-methadone, may improve the efficacy of morphine by attenuating the development of tolerance.