Human platelets express 2 G protein–coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y₁) via heterotrimeric guanosine 5-triphosphate (GTP)–binding protein G_q, and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y₁₂) via heterotrimeric GTP-binding protein G_i. Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y₁₂ receptor and are predicted to interact with the extracellular cysteine residues on the P2Y₁₂ receptor. In this study we identified the reactive cysteine residues on the human P2Y₁₂ receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y₁₂ receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC₅₀) values for ADP and 2-methylthio–ADP (2-MeSADP) when compared with the wild-type P2Y₁₂. Similarly, the median inhibitory concentration (IC₅₀) values for BzATP (2′,3′-O-(4- benzoylbenzoyl) adenosine 5′-triphosphate), an antagonist of the P2Y₁₂ receptor, also did not differ dramatically among these mutants and the wild-type P2Y₁₂receptor. pCMBS inactivated the wild-type P2Y₁₂ receptor in a concentration-dependent manner, whereas it had no effect on the P2Y₁ receptor. Finally, pCMBS partially affected the G_i coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y₁₂receptor–mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y₁ receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y₁₂ receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y₁₂ receptor, and thereby inactivate the receptor.