Previously, we showed that both D2 and D4 dopamine receptors inhibited adenylate cyclase in a pertussis toxin (Ptx)-sensitive manner in the dopamine-producing MN9D cell line, whereas only D2 receptors did so in a fibroblast cell line, CCL1.3. Of the known Ptx-sensitive G proteins, MN9D cells expressed G alpha i2, G alpha oA and G alpha oB, whereas CCL1.3 cells expressed only G alpha i2. Here we cotransfected MN9D and CCL1.3 cells with either the long form of the D2 receptor (D2L) or the D4 receptor and a mutant Ptx-resistant G protein alpha-subunit. When cotransfected CCL1.3 cell lines were tested for the ability of Ptx to block receptor-mediated inhibition of cyclic AMP accumulation, D2 receptors were found to couple to mutant G alpha i2 and G alpha i3 but not G alpha i1 or G alpha oA. D2 also coupled to mutant G alpha i2 but not G alpha oA in MN9D cells. In contrast, D4 receptors did not couple to either mutant G alpha i2 or G alpha oA subunits in MN9D cells. These data suggest that D4 receptor-mediated inhibition of adenylate cyclase is not coupled via the same mechanisms used by D2 receptors. D2L receptors are capable of coupling to more than one G protein in the modulation of cyclic AMP.