Adenosine 5'-O-(1-thiotriphosphate), Sp-diastereomer, is cyclized by adenylate cyclase from bovine brain to adenosine 3',5'-cyclic phosphorothioate, Rp diastereomer, establishing inversion of configuration for this reaction. This result can most easily be explained by a direct nucleophilic attack of the 3'-OH group on alpha-phosphorus without involving a covalent enzyme intermediate.