Cell extracts of the protozoan Tetrahymena thermophila contain high levels of glutathione S-transferase (EC 2.5.1.18). The level of the enzyme varied with the growth cycle, increasing in early stationary phase. It was localized in the cytoplasm. Only one major molecular form of the enzyme was detected by ion-exchange and gel filtration chromatography and isoelectric focusing (pI 7.0), and it was purified to homogeneity. The molecular weight of the enzyme was estimated to be 35,000 from gel filtration and 33,000 from sodium dodecyl sulfate-gel electrophoresis, indicating the enzyme is a monomer. The enzyme was specific for glutathione as the thiol substrate, and was most active with 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. It also exhibited glutathione peroxidase activity. The apparent Km values for glutathione and 1-chloro-2,4-dinitrobenzene were 3.2 and 1.5 mM, respectively. The enzyme catalyzed an S-aryltransfer with pentachloronitrobenzene (Km = 7.2 microM) consistent with the view that this pesticide is metabolized in part via S-conjugation pathways in T. thermophila.