1. Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenesis of C114 to serine. 2. The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ((-)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N-propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters 'a propyl cleft' on the receptor. The mutation hardly affected quinpirole-dependent [35S]-GTPgammaS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling. 3. N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4(2) receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4(2) receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4. We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4(2) receptor, is highly susceptible to external oxidizing agents.