The role of shoot and root glutathione (GSH) content and glutathione S-transferase (GST) activity in the response of the 'A632 × A635' and 'Anjou SC256' hybrids of maize (Zea mays L.) and of 'Jubilejnaja 50' wheat (Triticum aestivum L.) to the chloroacetanilide herbicide acetochlor was evaluated. The concentrations of root-applied acetochlor causing a 50% inhibition of plant shoot height were 20 μM for the tolerant 'A632 × A635' maize, 1 μM for the sensitive 'Anjou SC256' maize, and 0.1 μM for the very sensitive 'Jubilejnaja 50' wheat. The nonprotein thiol (mainly GSH) level in the roots of the tolerant 'A632 × A635' maize hybrid was 2-fold greater than that found in the roots of the sensitive maize hybrid and of wheat. Pretreatment with 10 μM of acetochlor induced the root nonprotein thiol levels of all three genotypes. The highest induction of root thiol content compared to controls was observed at 48 hr after acetochlor treatment and was 2.23-fold in the tolerant maize and 1.72-fold for the sensitive wheat. GST activities of etiolated maize and wheat seedlings were evaluated using both CDNB (1-chloro-2,4-dinitrobenzene) and [14C]-acetochlor as substrates. GST(CDNB) activity was greater in the roots than in the shoots of both maize hybrids. The shoot GST(CDNB) activity of both maize genotypes was similar, but the tolerant 'A632 × A635' maize had slightly higher root GST(CDNB) activity. In the sensitive wheat, similar shoot and root GST(CDNB) activities were observed. GST(CDNB) activity in roots of the maize hybrids and of wheat was enhanced by 70-100% at 48 hr after pretreatment with 10 μM of acetochlor. Shoot GST(CDNB) activity of maize or wheat was not induced significantly by acetochlor pretreatment. Root and shoot GST(acetochlor) activities of the maize hybrids and wheat were much lower than GST(CDNB) activities. Root GST(acetochlor) activities of the two maize hybrids were greater and more inducible by acetochlor pretreatment than those of the sensitive wheat. These results demonstrate the important role of endogenous levels of GSH and of GST activity in chloroacetanilide herbicide detoxication and selectivity. © 1991.