Changes in the composition and content of fatty acids (FA), including volatile fatty acids (VFA), in rumen fluid were analysed by a rumen-simulation technique (RUSITEC) following dietary supplementation with docosahexaenoic acid (C_22:6; DHA). Three different diets were tested: basal diet (CON, 60 : 40 forage to concentrate), basal diet plus 0.65% DHA (Trt1) and basal diet plus 1.30% DHA (Trt2). The experiment lasted 7 days (6 days for adaptation and 1 day for sampling). Culture fluid was collected every 2 h over a 12-h period on the last day of the experimental period. Compared to CON, the stearic (C_18:0) concentration decreased by 76.93 and 80.35% when Trt1 and Trt2 were administered, respectively (P<0.01). Whereas the trans-vaccenic acid (trans-11C_18:1; TVA) concentration increased by 185 and 126% compared to CON when Trt1 and Trt2 were administered, respectively, the cis-9, trans-11 conjugated linoleic acid (CLA) concentration increased by 111 and 142%. Compared to CON, addition of DHA changed the profiles of volatile fatty acids (VFA) in culture fluid, in which propionate content increase in replacement of acetate decrease. The concentrations of volatile fatty acids (VFA), TVA, and cis-9, trans-11 CLA were affected by the sampling time. These data indicate that dietary supplementation with DHA alters the VFA and FA content of culture fluid; however, these data should be replicated in vivo.