Rats fed n-3 fatty acid deficient diet through two generations exhibit decreased correct response ratios in a brightness-discrimination learning test. This is associated with a decreased docosahexaenoic acid (DHA) to arachidonic acid (AA) ratio in brain lipids. The altered learning behavior associated with a long-term n-3 deficiency was reversed by supplementing DHA after weaning only when the levels of competing n-6 fatty acids in the diet and brain lipids are limited. Endocannabinoid 2-arachidonoylglycerol (2-AG) levels were significantly elevated in the brain of n-3 deficient rats. We evaluated the effect of cannabinoid receptor CB1 antagonist SR141716A (SR) on the learning behavior. SR did not ameliorate the decreased learning performance in the n-3 deficient rats. However, the increased locomotor activity by n-3 deficiency was suppressed by SR. Therefore, the altered behavior is associated with, at least in part, the endocannabinoid signaling. We also found the decreased nerve growth factor (NGF) content and the increased level of the activated form of calmodulin kinase II in the hippocampus of n-3 deficient rats. DHA has an essential role in biomembrane structure and functions, which affect the cellular signaling related to learning behavior.