Low levels of docosahexaenoic acid (DHA) have been linked to a number of mental illnesses such as memory loss, depression and schizophrenia. While supplementation of DHA is beneficial in improving memory and cognition, the influence of dietary fats on the neurotransmitters and receptors involved in cognitive function is still not known. The aim of this study was to investigate serotonin receptor (5-HT(1A) and 5-HT2A), cannabinoid receptor (CB1) and gamma-aminobutyric acid type A (GABA(A)) receptor binding densities in the brain of male rats fed a high-saturated-fat (HF) diet, as well as the effect of DHA supplementation on HF diet. Alterations of these receptors in the post-mortem rat brain were detected by [(3)H]-WAY-100635, [(3)H]-ketanserin, [(3)H]-CP-55,940 and [(3)H]-muscimol binding autoradiography, respectively. In the hippocampus, the 5-HT(1A), CB1 and GABA(A) receptor binding densities significantly increased in response to an HF diet, while in the hypothalamus, 5-HT(1A) and CB1 binding densities significantly increased in HF-fed rats. Importantly, DHA supplementation prevented the HF-induced increase of receptors binding density in the hippocampus and hypothalamus. Furthermore, DHA supplementation attenuated 5-HT2A receptor binding density in the caudate putamen, anterior cingulate cortex and medial mammillary nucleus, which was also increased in HF group. This study showed that an HF diet increased 5-HT(1A), 5-HT2A, CB1 and GABA(A) receptor binding densities in the brain regions involved in cognitive function and that dietary DHA can attenuate such alterations. These findings provide insight into the mechanism by which DHA supplementation ameliorates reduced cognitive function associated with an HF diet.