Differences in visual, auditory, and learning tasks have been reported for infants and animals given diets varying in omega-3 fatty acids, but the neurobiochemical basis for these changes is unclear. This study investigated the effect of feeding formula with 0.8% energy C18:2 omega-6 + 0.05% C18:3 omega-3 (low), or 8.3% C18:2 omega-6 + 0.8% C18:3 omega-3 (adequate), with and without 0.2% energy arachidonic acid (C20:4 omega-6) and 0.16% docosahexanoic acid (C22:6 omega-3), on monoaminergic neurotransmitters in different brain regions of piglets fed formula from birth to 18 d. The amount of C18:2 omega-6 + C18:3 omega-3 fed in formula had a significant effect on frontal cortex dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindolacetic acid; striatum serotonin and inferior colliculus serotonin, resulting in lower concentrations in piglets fed the low compared with adequate C18:2 omega-6 + C18:3 omega-3 formula. Inclusion of arachidonic acid and docosahexanoic acid in the low, but not in the adequate, C18:2 omega-6 + C18:3 omega-3 formula resulted in increased concentrations of all monoamines in the frontal cortex, and in striatum and inferior colliculus serotonin. Feeding arachidonic acid and docosahexanoic acid in the formulas increased dopamine and 5-hydroxyindolacetic acid in superior and inferior colliculus, areas related to processing and integration of visual and auditory information. Higher dopamine and 5-hydroxyindolacetic acid were found in these regions even when arachidonic acid and docosahexanoic acid were added to the C18:2 omega-6 + C18:3 omega-3 adequate formula. This study suggests that functional changes among animals and infants fed diets varying in omega-6 and omega-3 fatty acids may involve altered neurotransmitter metabolism.