14C-chlorsulfuron {2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino] carbonyl] benzenesulfonamide} and its metabolites were extracted from flax (Linum sativum L.), field penny cress (stinkweed) (Thlaspi arvense L. # THLAR), and wheat (Triticum aestivum L.) with an average efficiency of 94% using an aqueous extraction procedure. Chlorsulfuron and its metabolites were separated on a reverse-phase preparative chromatography column by eluting with a step gradient of aqueous 0.1% (v/v) formic acid and methanol. Major peaks were eluted at 35 and 45% methanol, and minor peaks at 10, 25, and 100% methanol. The 45% methanol peak represented unmetabolized chlorsulfuron or its hydroxylated derivative and was the only fraction that exhibited biological activity. In wheat and flax, 75 and 62%, respectively, of the extracted activity were eluted by 35% methanol and, in the case of wheat, this fraction was shown to be a glycosylated derivative of chlorsulfuron. Although 14C-chlorsulfuron was metabolized to a lesser extent in field pennycress, our data indicate that over 50% of the absorbed 14C-activity was recovered in forms other than 14C-chlorsulfuron 5 days after treatment. The separation procedure can be used readily to assess the amount of chlorsulfuron detoxification that occurs in plants.