Glutathione S-transferase (GST), glutathione peroxidase (GPX), and glutathione reductase (GR) are enzymes that utilize glutathione to play an important role in plant defense mechanisms. In leafy spurge (Euphorbia esula L.), transcript and activity profiles for these enzymes are differentially influenced in tissue exposed to xenobiotic (diclofop-methyl) and environmental stress (cold and drought). Five different EeGST cDNA (including phi, tau, theta, and zeta class GSTs), one EeGPX cDNA, and one EeGR cDNA showed differential expression patterns in leafy spurge plants exposed to diclofop-methyl-, cold- and drought-stress. Tissue treated with diclofop-methyl also had increased GST, GPX and GR activities that were preceded or paralleled by increased gene expression. Transcript profiles resulting from drought-stressed plants were similar to transcript profiles from diclofop-methyl-treated plants but not cold-stressed plants. GPX activity in leafy spurge protein extracts was not bound to either S-hexylglutathione- or glutathione-agarose columns but instead co-migrated with fractions of GST activity that also were not bound by affinity chromatography. Fractions of GST proteins that were bound to S-hexylglutathione revealed that increased GST activity in diclofop-methyl-treated tissue could be identified as phi- and tau-type GSTs.