Zinc (II) modulates the function of many integral membrane proteins. To identify the Zn(2+)-binding site responsible for allosteric modulation of the D(2) dopamine receptor, we first demonstrated that the binding site is likely located in extracellular loops or in transmembrane regions that are accessible from the extracellular milieu. We mutated every histidine in these regions to alanine; two mutants, H394A and H399A, exhibited a reduced response to Zn(2+). Combined mutation of H394 and H399 caused a larger effect of zinc than did either single mutation. Mutation of other potential Zn(2+)-binding residues predicted to be in proximity to H394 or H399 did not substantially alter the potency of Zn(2+). The double mutant H394A/H399A was similar to D(2) in affinity for [(3)H]spiperone and ability to inhibit cyclic AMP accumulation. We conclude that binding of Zn(2+) to H394 and H399 on the dopamine D(2) receptor contributes to allosteric regulation of antagonist binding.