Peramine, a novel fungal metabolite isolated from perennial ryegrass (Lolium perenne L.) infected with the fungal endophyte Acremonium lolii Latch, Christensen, Samuels, is a highly active feeding deterrent to the Argentine stem weevil (Listronotus bonariensis Kuschel) and implicated in the resistance of endophyte-infected ryegrasses and fescues to this pest. The original isolation from herbage required final purification as the diacetyl derivative and was impractical for producing sufficient underivatized material for further biological testing. We now report an improved procedure for the isolation of peramine from seed using ion exchange and reversed-phase chromatography, taking advantage of its strongly basic guanidino group and lipophilic pyrrolopyrazine moieties. This procedure yielded pure crystalline peramine for the first time, and spectroscopic data (hrms, ¹H nmr, ¹³C nmr) supports the 3-(3'-guanidinopropyl)-2-methylpyrrolo[1,2-a]pyrazin-1(2H)-one structure originally proposed from the nmr analysis of the diacetyl derivative. TLC analysis of A. lolii infected seeds and seedlings indicated peramine was present in greater or comparable amounts to herbage, with relatively less pigments making seeds a preferred source. The new procedure involves: crude EtOH extract from ryegrass seeds defatted with petroleum ether, sequential passage through Dowex 2 X 8 (OH⁻ form) and Amberlite CG-50 (H⁺ form) columns in series, elution from Amberlite with HCOOH, concentration, desalting and further purification on C-18 reversed-phase silica, precipitation as picrate, and recrystallization as sulfate or hydrobromide. Spectroscopic data (hrms confirming molecular formula C₁₂H₁₇N₅O for the free base, ¹H nmr with connectivities and coupling constants matching diacetyl peramine, ¹³C nmr with resonances consistent with the proposed structure) confirmed the structure, with some revisions to previous assignments for diacetyl peramine. Biological activity tests showed peramine deterred feeding by adult and larval Argentine stem weevil at concentrations down to 0.1 and 2 pg/g, respectively, and disrupted development of the cutworm Graphania mutans Walker at 10 pg/g; further studies on its biological activity are continuing.