One of the sources of variation in the in vitro gas production (GP) technique is the inoculum source that is used and this could be greatly affected by the diet that is given to the donor animal. To study the effect of the host diet on the gas production profiles of grass hay and high temperature dried grass, two cows were offered either a silage: barley diet (80:20 dry-matter (DM) basis; GSB) or barley straw (ad libitum; STR). An adaptation period of 3 weeks was used and each animal experienced each diet type twice. Rumen fluid and solids were collected at the end of each 3-week period and used to inoculate the substrate cultures. The volume of gas produced (mllg DM) was 379 and 289 for GSB and STR respectively; gas yield (mllg organic matter degraded) was 442 and 411. The maximum fractional rates of degradation (per h) were 0·067 and 0·061 and the time (h) taken to reach these rates were 5·3 and 12·6. None of these differences was significant. There were also no significant differences in the concentration of total volatile fatty acids (VFA) in the final incubation media, or in the molar proportions of individual VFA. These results suggest that the fermentation stoichiometry was not affected by donor animal diet and, while microbial activity from STR was lower, this did not significantly affect the GP profile. The difference in microbial activity between the two diets was perhaps minimized by taking the sample of rumen contents before the morning meal.