Considerable efforts have been made regarding the use of faecal material to provide a microbial inoculum for in vitro feed evaluation systems. However total gas production, rate of gas release and the extent of degradation of feeds incubated using faecal inoculum are lower than those incubated in a rumen fluid medium. It has been suggested that this is due to lower microbial activity, a consequence of the different microflora and reduced microbial numbers (e.g. Mauricio, 1999). Microbial populations are dynamic so, as their enzyme activity profiles change rapidly, little information is obtained from examining these. However, their hydrolytic activity as reflected by their ability to degrade specific substrates can be simply measured and provides a potential method with which to assess the quality of inocula with respect to their use in in vitro systems. The data presented here are from a larger study in which the differences between the hydrolytic activity of faecal material and rumen contents as influenced by the time of sampling were assessed in vitro.