Glutathione S-transferase (RX: glutathione R-transferase, EC 2.5.1.18) from human placenta has been purified to homogeneity. This enzyme, transferase pi, is an acidic protein (isoelectric point at pH 4.8) composed of two subunits. The molecular weights for the dimer and monomer were determined by independent methods as 47,000 and 23,400, respectively. These properties are not significantly different from those of glutathione S-transferase rho from human erythrocytes. Antibodies to transferase pi reacted with the enzyme from erythrocytes but not with the basic transferases alpha - epsilon and the neutral transferase mu isolated from human liver. Antibodies to the latter enzymes did not react with the transferase from placenta. Further similarities between transferases pi and rho appear in amino acid compositions, kinetic constants and substrate specificities. Both the placental and the erythrocyte enzyme have considerably higher activity with ethacrynic acid than any other of the human glutathione S-transferases. The glutathione S-transferase could be distinguished from two additional acidic glutathione-dependent enzymes, glyoxalase I and selenium-dependent glutathione peroxidase. It is concluded that transferase pi from placenta is identical with or very closely related to transferase rho from erythrocytes.