Many class C GPCRs function as homo- or heterodimers and several class A GPCRs have also been shown to form a homodimer. We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under reducing and nonreducing conditions. Under nonreducing conditions, approximately half of the P2Y2Rs were electrophoresed as a dimer. We then produced Cys to Ser mutants at four sites (Cys25, Cys106, Cys183, Cys278) in the extracellular domains of P2Y2R and examined the effect on dimer formation and receptor activity. All single mutants formed dimers similarly to the wild-type protein, but C25S, C106S and C183S P2Y2R lost activity, while C278S P2Y2R maintained weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required for monomer activity, but is not needed in one protomer for homodimer activity; and Cys278 can be replaced in the monomer and homodimer. Approximately half of C25S/C278S double mutants were electrophoresed as a dimer, similarly to the wild-type and single mutants, and dimers with the wild-type protein were active. These results suggest involvement of Cys106 and Cys183 in disulfide bonding between protomers in homodimer formation.