To investigate the effect of dietary docosahexaenoic acid(DHA) and environmental enrichment on brain fatty acid composition and acetylcholinesterase(AChE) activity, two groups of was fed isocaloric diets containing 10 or 12% dietary lipids for 7 weeks. A third group was fed 10% (w/w) dietary lipids with supplemented 2% DHA-rich fish oil. Each diet group was housed either in a stainless steel cage individually or in a large enriched cage with toys where 7 rats were kept together. The fatty acid composition of plasma and brain was significantly affected by dietary lipid composition but not by environmental enrichment. Fish oil supplementation significanlty decreased plasma levels of monounsaturated fatty acids(MUFA) and increased polyunsaturated fatty acids(PUFA). Fish oil supplemented groups also maintained lower plasma n-6 fatty acids and higher n-3 fatty acids levels than unsupplemented groups. The fish oil supplementation significantly decreased arachidonic acid and increased eicosapentaenic, docosapentaenoic acids, and DHA in brain fatty acid composition. In addition, brain DHA level in supplemented groups tended higher than the unsupplemented. Brain, AChE activity significantly increased by the environmental enrichment but not by the fish oil supplementation. These finding suggest that the 2% fish oil (0.57% DHA & 0.31% EPA, per diet weigth) supplementation is enough to accumulate n-3 fatty acids and to change the n-6 n-3 ratio in brain and environmental enrichment might promote the learning ability.