The glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of dimeric proteins that catalyze reactions between glutathione (GSH) and various electrophiles. A partial cDNA for human GST pi was obtained and the open reading frame completed. The completed cDNA was cloned, and GST pi protein was expressed in bacteria. Cloned enzyme was purified and had the same kinetic constants, molecular mass, pI value, and N-terminal sequence as placental GST pi except that some of the polypeptides had N-terminal methionines. A radiolabeled azido derivative of GSH, S-(p-azidophenacyl)-[3H]glutathione, was used to photoaffinity-label the active site of the cloned enzyme. Labeled enzyme did not bind to a GSH-agarose affinity column. Labeling was prevented in the presence of S-hexylglutathione, and noncovalently-bound azido affinity label was a competitive inhibitor towards 1-chloro-2,4-dinitrobenzene and GSH. These results suggest that the azido label was binding at the active site of the enzyme. Photoaffinity-labeled enzyme was trypsinized, and two labeled peptides were purified and sequenced. One peptide corresponded to residues 183-188, whereas the other corresponded to residues 183-186. These residues appear to form part of the hydrophobic (H-site) binding region of human GST pi that has not been shown previously. Cloned enzyme was subjected to radiation inactivation to assess the importance of subunit interactions in the maintenance of catalytic activity. The target size of enzymatic activity (23 kDa) was not significantly different from that of the protein monomer (24 kDa). Therefore, each subunit of human GST pi appears to be capable of independent catalytic activity.