The effect of Mg2+ binding on the conformation of the inactive GDP- bound complex of the heterotrimeric G protein α subunit G(iα1) has been investigated by X-ray crystallography. Crystal structures of the G(iα1) · GDP complex were determined after titration with 5, 10, 100, and 200 mM Mg2+. Comparison of these structures with that of the Mg2+-free complex revealed Mg2+ bound at the same site as observed in the structure of the active, G(iα1) · GTPγS · Mg2+-bound complex of G(iα1), with a similar coordination scheme except for the substitution of a water molecule for an oxygen ligand of the γ-phosphate of G(iα1) · GTPγS · Mg2+. In contrast to the GDP · Mg2+ complex of G(tα) and of other G proteins, switch I residues of G(iα1) participate in Mg2+ binding and undergo conformational changes as a consequence of Mg2+ binding. Partial order is induced in switch II, which is disordered in the Mg2+-free complex, but no order is observed in the switch III region. This contrasts with the GDP · Mg2+ complex of G(tα) in which both switch II and III switch are ordered. Mg2+ binding also induces binding of an SO42- molecule to the active site in a manner which may mimic a G(iα1) · GDP · PO42- · Mg2+ product complex. Implications of these findings are discussed.