Two in vitro experiments were conducted to determine the effect of freezing on the fermentative activity of equine faecal inocula for use in an in vitro gas production technique. In Experiment 1, high-temperature dried alfalfa (HTA) and mature grass hay (GH) were incubated with an inoculum prepared either fresh (0 d) or following storage at -20 degrees C for 7 days (7 d). In Experiment 2, HTA, GH, high-temperature dried grass (HTG), and unmolassed sugar beet pulp (SB) were incubated with an inoculum prepared either fresh (0 d) or following storage at -20 degrees C for 24 hours (24 h), 48 hours (48 h) or 72 hours (72 h). Substrate/inocula combinations were fermented using an in vitro gas production (GP) technique. In both experiments, analysis of cumulative gas production values revealed an interaction (P<0.05) between inocula and substrate. Gas production from bottles containing HTA as the substrate was less affected by inocula source (fresh or frozen; 7 d, 24 h, 48 h and 72 h) compared to all other substrates. In Experiment I, total GP was reduced for GH inoculated with frozen faeces (7 d) compared to fresh faeces, and unaffected for HTA. In Experiment 2, total GP was also unaffected by inocula source in bottles containing HTA, whilst reduced in those containing HTG and SB and HTA, respectively when inoculated with frozen (24 h) compared to fresh faeces. However, the rate of gas production was also affected by inocula source in both experiments across all substrates. In conclusion, preservation of equine faecal inocula by freezing for 7 d or 24, 48 or 72 h has a marked effect on the extent and rate of substrate fermentation in vitro. Moreover, the extent of these effects appears to be dependent upon the nature of substrate used in the in vitro incubations. Consequently, further work is required on the use of frozen faeces as inocula for the in vitro gas production technique before this preservation treatment can be used routinely as an alternative to using fresh faeces. (C) 2012 Elsevier B.V. All rights reserved.