Ergovaline (EV), the primary alkaloid implicated as a causative agent in fescue toxicosis, was extracted from endophyte-infected tall fescue seed (E+), and extraction procedures were developed to purify EV since it is not commercially available. The E+ seed was extracted with 5% lactic acid, initially cleaned up using liquid-solid extraction with Bio-Beads SM-2, and further purified using reversed-phase C-18 HPLC with a gradient of acetonitrile, 2.6 mM ammonium carbonate, and methanol. Incubation of purified ergovaline with rat brain homogenates resulted in a dose-dependent inhibition of Na+/K+ ATPase activity, with 50% inhibition occurring at 50 µM concentration of EV. Mg2+ activated ATPase was not significantly affected by the same treatment using doses up to 200 µM EV. Kinetic data indicated that EV interacted (uncompetitively) and inhibited synaptosomal Na+/K+ ATPase activities. This study demonstrates that EV isolated from E+ fescue seed can alter brain Na+/K+ ATPase activity and may be a contributing causative factor in fescue toxicosis.