Horseweed (Conyza canadensis) is a cosmopolitan weed that commonly grows throughout North America. Horseweed that is not completely controlled by normal applications of glyphosate has been reported in western Tennessee. This research had three objectives: (1) to develop and validate an analytical procedure for the quantitative determination of shikimate, an important indicator of glyphosate activity in plants; (2) to confirm resistance to glyphosate in a horseweed population; and (3) to examine the accumulation of shikimate in both glyphosate-resistant and glyphosate-susceptible horseweed plants. The analytical procedure to determine shikimate used extraction with 1 M HCl for 24 h, followed by liquid chromatography using photodiode array detection, and shikimate recoveries were >or=82%. Glyphosate applications of both 0.84 kg ae/ha (the standard application rate) and 3.8 kg ae/ha to susceptible plants caused complete plant death. The same glyphosate applications to putative resistant populations caused less than 15% growth reduction as determined by visual evaluations, and fresh weights of these resistant plants 17 days after glyphosate treatment (DAT) were reduced an average of 45% in one population and were not affected in a different population. This direct comparison conclusively confirms that horseweed plants collected in western Tennessee in 2002 are resistant to 4 times the normal application dosage of glyphosate. The glyphosate-resistant horseweed biotypes still exhibited some herbicidal effects from the glyphosate, such as yellowing in the most actively growing, apical shoot meristems. The yellowing in the shoot apexes was transitory, and the plants recovered from this damage. Shikimate concentrations in all untreated horseweed plants were less than 100 microg/g, which was significantly less than that in all plants which had been treated with 0.84 kg ae/ha of glyphosate. Unexpectedly, shikimate accumulated (>1000 microg/g) in both resistant populations and the susceptible population. However, there were differences in shikimate accumulation patterns between resistant and susceptible horseweed biotypes. Shikimate concentrations in resistant populations declined about 40% from 2 to 4 DAT, while shikimate concentrations in the susceptible horseweed plants increased about 35% from 2 to 4 DAT. The confirmed resistance of a widespread weed implies that alternative control strategies for glyphosate-resistant horseweed will be needed in those no-tillage production systems where it commonly occurs.