The D(2) dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype-selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120-fold) for the D(4) subtype of dopamine receptor were tested on wild-type and mutant D(2) receptors. Five of the selective ligands were observed to have 21-fold to 293-fold increases in D(2) receptor affinity when three non-conserved amino acids in TM2 and TM3 were mutated to the corresponding D(4) amino acids. The two ligands with the greatest improvement in affinity for the D(2) mutant receptor [i.e., 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine (L-750,667) and 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine (RBI-257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild-type receptor, concentrations of L-750,667 or RBI-257 that produced large reductions in the potency of quinpirole's functional response in the mutant did not significantly reduce quinpirole's functional response in the wild-type D(2) receptor. In contrast to RBI-257 which is an antagonist at all receptors, L-750,667 is a partial agonist at the wild-type D(2) but an antagonist at both the mutant D(2) and wild-type D(4) receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D(2) dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non-conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype-selective ligands and the wild-type D(2), mutant D(2), or wild-type D(4) receptors.