AbstractPrevious work demonstrated two high‐affinity PCP binding sites in guinea pig brain labeled by [3H]TCP (1‐{1‐[2‐thienyl]cyclohexyl}piperidine): site 1 {N‐methyl‐D‐aspartate [NMDA]‐associated} and site 2 {dopamine‐reuptake complex associated}. The present study examined brain membranes prepared from various species, including human, for the presence of site 2, defined as binding in the presence of (+)‐5‐methyl‐10, 11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐imine maleate ((+)‐MK801) minus binding in the presence of 10 μM TCP (nonspecific binding). Studies were conducted in absence of sodium which was found to be inhibitory to [3H]TCP binding. The results demonstrated detectable levels of site 2 in brain membranes of guinea pig, rabbit, pig, mouse, sheep, and human but not in the rat or chicken. Using human cortical membranes, site 2 was the predominant binding site. Detailed studies conducted with human cortical tissue showed that high‐affinity dopamine {1‐[2‐]bis(4[fluorophenyl)[methoxy]ethyl]4‐(3‐phenylpropyl)piperazine (GBR12909)}, [1, 2]benzo(b)thiophenylcyclo‐hexylpiperidine (BTCP), and serotonin (fluoxetine) uptake inhibitors produced a wash‐resistant inhibition of [3H]TCP binding to site 2, but not site 1. Preincubation of guinea pig brain membranes with BTCP was shown to produce an increase in the dissociation rate of [3H]TCP from PCP site 2. Structure activity studies with various uptake inhibitors showed that GBR12909, benztropine, fluoxetine, and BTCP have higher affinity for site 2 than for site 1. (+)‐MK801, ketamine, and tiletamine were very selective for site 1, whereas dexoxadrol and TCP were moderately selective for site 1. These results suggest that human cortex possesses high‐affinity PCP binding sites associated with biogenic reuptake binding sites, and that guinea pig brain, but not rat brain, may be an appropriate animal model for studying PCP site 2 in human brain.