The objective of this study was to examine the effect of ensiling and maturity on the in vitro digestion kinetics of the perennial ryegrass cell wall fraction. Preliminary methodological studies concluded that (i) in vitro cell wall digestion profiles were optimised when fermentation tubes were horizontally incubated, (ii) perennial ryegrass cell wall isolation by neutral detergent extraction but not by aqueous extraction (70 °C) adversely affected in vitro digestion kinetics (iii) method of inoculum preservation (untreated and frozen at - 20 °C, with or without cryoprotectant, with or without pre-incubation) did not affect the rate but all imposed a lag (p<0.05) and altered the extent of dry matter (DM) digestion, when compared with fresh inoculum. Pre-incubation was beneficial in the absence of a cryoprotectant only (p<0.05) and the digestion kinetics of the frozen un-treated inoculum were similar to preservation with a cryoprotectant. A dual flow semicontinuous culture was established. In vitro protozoal numbers were less than in vivo (p<0.001) and in vivo ruminal diurnal trends for volatile fatty acid (VFA), ammonia and lactate were qualitatively simulated. When the fresh forage was incubated in vitro, ensiling reduced (p<0.001) the apparent extent of digestion (AED) of a late season perennial ryegrass cell wall fraction. Ensiling had no effect on the AED of the fractionated cell wall fraction, removed from the whole forage by aqueous extraction. There was a maturity x forage interaction for the cell wall digestion of fresh (p<0.01) and fractionated (p<0.05) perennial ryegrass ensiled at different maturities. Maturity (p<0.001) but not ensiling adversely affected the digestion of the isolated cell wall fraction. Ensiling per se decreased the microbial protein production (p<0.001) from the watersoluble fraction but did not affect VFA production. The AED of the isolated cell wall fraction from an extensively preserved perennial ryegrass forage was increased when supplemented with the water-soluble component of the fresh herbage (WG) (p<0.05) or with WG and nitrogen (p<0.05). The AED of the isolated cell wall fraction from the restrictively preserved forage was not influenced by supplementation. The biochemical alterations in the Wg fraction due to ensiling did not influence cell wall digestion of the fresh or extensively preserved forage nor did it influence protozoal numbers, microbial protein or VFA production in the rumen semi-continuous culture.