Timothy, harvested at the seed setting stage, was subjected to 3 different treatments: (1) ball milling followed by in vitro rumen digestion, (2) ball milling followed by cellulase digestion, (3) digestion by a heifer, and then dioxane-soluble lignins (RDL, CDL and HDL, respectively) were isolated from the 3 resultant residues. RDL contained a larger amount of lignin components and smaller amounts of carbohydrate residues, and bound phenolic acids than CDL and its composition resembled that of HDL. Alkaline nitrobenzene oxidation and IR spectra showed that all the lignins consisted of guaiacyl-syringyl lignins. The molar ratio of syringaldehyde to vanillin was higher for HDL than for CDL and RDL. In gel permeation chromatograms, RDL and HDL were distributed in lower molecular weight regions than CDL. The results suggested that timothy lignin was depolymerized and the phenolic acids bound to timothy lignin disappeared by rumen fermentation.