To investigate effects of hay intake and feeding sequence on indicators of the microbial activity within the hindgut, six horses were fed 1.00 kg oats plus 0.50, 0.67, 0.83 or 1.00 kg hay/100 kg body weight (BW) · day, each for 14 days. Oats was offered either 30 min prior to hay (OA) or in the reversed sequence (HA) in a 2 · 8-week crossover design. Because typically exercised horses should be subjected to the study, faeces was used as substrate. Faecal dry matter (DM), the faecal watersÕ short-chain fatty acids (SCFA, in mmol/l) and molar percentages (mol%) of propionate and iso-butyrate were highest with OA (p < 0.01). Acetate mol%, acetate-propionate quotient (A/P) and buffering capacities 1 and 2 (BC1: current pH to pH 6; BC2: pH 6 to 5) of the faecal water were highest with HA (p < 0.01). While the hay intake rose, faecal pH, acetate mol%, A/P, BC1 and BC2 (the latter with HA only) increased (p < 0.05), but DM, SCFA and propionate mol% declined (p < 0.05). The hay-induced rise in A/P and BC1 was much higher with HA than with OA. l-Lactate and ammonia were unaffected by the feeding sequence and hay intake. In conclusion, hay intake and feeding sequence influence the microbial activity within the hindgut, although the concentrate level remains consistent. In horse rations with 1.00 kg oats/100 kg BW · day amounts of at least 0.83 kg hay/100 kg BW · day and offering the hay first seem to have the potency to protect the hindgut content from acidification. Behavioural abnormality was not observed any longer first with 1.00 kg hay/100 kg BW · day.