A novel radioiodinated ligand with a high specific activity (2,200 Ci/mmol), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine ([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 +/- 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(-)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [125I]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contrast to [125I]S(-)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPgammaS binding further demonstrated the antagonist property of IPMPP.