The resistance mechanism of vetiver (Chrysopogon zizanioides Nash) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases (GST) and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the role of glutathione-S-transferase in the detoxification of atrazine, as determined in vetiver leaf and root of 5-week- and 8-month-old plants grown in hydroponics. Fresh vetiver roots and leaves were cut to extract and study their GST activities toward 1-chloro-2,4-dinitrobenzene (CDNB) and atrazine, using HPLC to quantify the biosynthesis of atrazine conjugates. The global GST activity was three orders of magnitude higher than that of GST isoform able to conjugate atrazine. In vitro activities of conjugation of CDNB were similar in all root and leaf vetiver extracts, whereas activities on atrazine were only detected in leaf extracts. Entire vetiver plants exposed to 14C-atrazine were found to accumulate radioactivity at the tip of leaves under moderate transpiring conditions (75% humidity). Vetiver transformed atrazine mainly into polar compounds, identified as conjugates by TLC analyses. After 20 days of exposure, the proportion of atrazine and metabolites compared to the total penetrated radioactivity into plants was: 50% of conjugates, 28% of atrazine plus possible dealkylates and 22% of unidentified products. The maximum conjugates production was observed at the tip of leaves (29 nmol g−1 fresh biomass), as compared to roots (6 nmol g−1 fresh biomass). Altogether, these metabolic features indicate that conjugation to glutathione was a major metabolic pathway to detoxify atrazine in vetiver.