The dopaminergic antagonist haloperidol also acts at the psychotomimetic sigma receptor and has been proposed to act at N-methyl-D-aspartate (NMDA) receptors. We have investigated the basis of its action at NMDA receptors using native and recombinant receptors and as a function of brain development. Haloperidol inhibits binding of 125I-(+) MK 801 to NMDA receptors formed from NR 1a/2B with high affinity but has lower affinity at receptors formed from NR 1a/2A. No differences are noted between different NR 1 splice variants transfected with NR 2B. Differences are also seen between neonatal receptors and adult receptors; neonatal receptors are inhibited by haloperidol with an IC50 value that is 50 times lower than that of adult receptors. Other agents acting at the sigma receptor inhibit binding of 125I-(+) MK 801 to NMDA receptors, and are slightly more potent at neonatal receptors. However, the rank order of potency for sigma agents at sigma receptors does not match their rank order of potency at NMDA receptors, and the prototypic sigma ligand ditolylguanidine shows no preference between NR 1a/2A and NR 1a/2B receptors. This suggests that haloperidol exerts its effects on the NMDA receptor at a distinct site from the pharmacologically defined sigma receptor. The effects of haloperidol are also modified by spermidine noncompetitively, which differs from the effects of spermidine on ifenprodil-mediated inhibition of NMDA receptors. This suggests that haloperidol interacts with NMDA receptors at a site distinct from the polyamine or ifenprodil sites.