Previous investigators have identified a number of ergot alkaloids (EAs) in tall fescue (Festuca arundinacea Schreb.) infected by the endophytic fungus, Neotyphoidium coenophialum [(Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.]. Their results, however, may have been confounded by the presence of the related parasitic ergot fungus (Claviceps purpurea [Fr.:Fr.] Tul.), which also produces EAs. Semipreparative HPLC was used to separate fractions giving a high fluorescence response in a sample of endophyte-infected (EI) tall fescue seeds, carefully examined to eliminate the possibility of Claviceps infection. Analytical high-performance liquid chromatography (HPLC) and LC/electrospray ionization mass spectrometry (ESI-MS) were used to identify EAs after isolation. Clearly identified in the spectra were ergine, ergovaline, ergosine, ergonine, and previously undescribed EAs, didehydroergovaline and aci-ergovaline, including their epimers. Several additional EAs may have been present, but structures could not be confirmed by their spectra. Not found in the isolated fractions were ergonovine, ergotamine, or lysergylmethylcarbinolamide. The developed HPLC method was used to determine the alkaloids in plants, culms, and seeds of EI tall fescue.