Docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids are n-3 polyunsaturated fatty acids with a therapeutic potential for CNS diseases. Here, using an in situ brain perfusion technique in mice, we show that [(14)C]-DHA and [(14)C]-EPA readily cross the mouse blood-brain barrier (BBB) with brain transport coefficients (Clup) of 48+/-3microlg(-1)s(-1) and 52+/-4microlg(-1)s(-1), respectively. Mechanical capillary depletion of brain homogenates showed that less than 10% of [(14)C]-DHA or [(14)C]-EPA remained in endothelial cells of the brain vasculature, demonstrating that both molecules fully crossed the BBB. Addition of bovine serum albumin decreased the Clup of [(14)C]-DHA to 0.6+/-0.3microlg(-1)s(-1), indicating that binding to albumin reduced importantly, but not totally, the passage of DHA through the BBB. The Clup of [(14)C]-DHA or [(14)C]-EPA was not saturable at concentration up to 100microM, suggesting that these compounds crossed the BBB by simple diffusion. However, long-term high-DHA dietary consumption reduced the Clup of [(14)C]-DHA to 33+/-6microlg(-1)s(-1) (-20%, p<0.01). These results confirm that the brain uptake of DHA or EPA perfused with a physiological buffer is comparable to highly diffusible drugs like diazepam, and can be modulated by albumin binding and chronic dietary DHA intake.