||Imidacloprid, [1-(6-chloro-3-pyridylmethyl)-2-nitroimino-imidazolidine], is a second generation neonicotinoid pesticide that has been used to treat plants against threatening pests. In the years 2005 and 2006 the U.S. Department of Agriculture, U.S. Forest Service treated over 100 eastern hemlock trees with imidacloprid against the Hemlock Woolly Adelgids. We developed a method to quantify the concentration of imidacloprid present in the hemlock xylem fluid using derivatization gas chromatography-mass spectrometry (GC/MS). Using ELISA, imidacloprid levels in 4811 samples of hemlock xylem fluid were measured and a subset of samples was compared to levels measured by GC/MS. A comparison of the GC/MS and ELISA methods indicated that significant cross-reactivity is present in the imidacloprid levels of hemlock xylem fluid samples using the ELISA method. The study coordinated by the U.S. Forest Service measured the uptake, persistence, and mobility of imidacloprid in treated hemlock trees in a natural setting. ELISA was used to determine the levels of imidacloprid by two different application techniques; soil injection and trunk injection as well as the determination of imidacloprid levels spatially throughout different areas of the tree and temporally over subsequent seasons was also determined using ELISA. Results indicated that a trunk injection during the spring season is the most efficient route for uptake of imidacloprid into the hemlock xylem fluid.