To investigate the roles of individual transmembrane segments (TM) of the human D3 dopamine receptor in its ligand-receptor interactions, we generated chimeric receptors in which its TMs were replaced, one at a time, partially or entirely, by the corresponding TM of the homologous human D1 receptor. Ligand binding properties of the chimeras, as expressed heterologously in Sf9 cells using recombinant baculoviruses, indicate that the critical binding regions for D3-selective (over D1) ligands reside at narrow regions (6 to 8 residues) near the extracellular surface for TMI, II, IV and VI, while TMV seems to be minimally involved in the ligand selectivity. For TMIII and TMVII, the critical regions seem to be deeper, involving at least the 10 residues near the extracellular surface for TMIII, and the entire TM segment for TMVII. This is based on our current observations that the chimeras with the D3 sequence in the critical regions, although the rest of the TM is of D1 origin (except TMVII), showed the binding properties indistinguishable from those of the wild-type receptor. The chimeras with the D1 sequence in the regions, on the other hand, showed ligand binding characteristics wildly variable depending on substituted TMs: Most marked decreases in ligand affinities were observed with the chimeras of TMIII and VII, and intermediate changes with those of TMIV and VI. Replacements of TMV produced no appreciable effects on the affinities of 14 test ligands (except for one). The chimeras of TMI and II with the D1 sequence in the critical regions showed no appreciable specific binding for several radioactive D3-selective ligands, possibly reflecting their critical roles in assembly and folding of the receptor. These critical regions of the D3 receptor were highly homologous to those of the D2 receptor, except for several nonconservatively substituted residues, which could be exploited to develop ligands selective for the D3 over D2 dopamine receptor or vice versa.