In the brain, polyunsaturated fatty acids (PUFA), especially arachidonic acid and docosahexaenoic acid (DHA), are required for regulating membrane fluidity, neuronal survival and signal transduction. Since the brain cannot synthesize n-6 and n-3 PUFA de novo, they must be supplied from the blood. However, the methods of PUFA entry into the brain are not agreed upon. This study tested the necessity of CD36, a candidate transporter of unesterified fatty acids, for maintaining brain PUFA concentrations by comparing brain PUFA concentrations in CD36(-/-) mice to their wild-type littermates. Because CD36(-/-) mice have been reported to have impaired learning ability, the PUFA concentrations in different brain regions (cortex, hippocampus, cerebellum and the remainder of brain) were investigated. At 9 weeks of age, the brain was separated into the four regions and fatty acid concentrations in total and phospholipid classes of these brain regions were analyzed using thin layer and gas chromatography. There were no statistical differences in arachidonic acid or DHA concentrations in the different brain regions between wild-type and CD36(-/-) mice, in total or phospholipid fractions. Concentrations of monounsaturated fatty acids were decreased in several phospholipid fractions in CD36(-/-) mice. These findings suggest that CD36 is not necessary for maintaining brain PUFA concentrations and that other mechanisms must exist.