Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [14C]octanoic and [14C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [14C]linoleic acid was not probenecid sensitive. With 0.5 microM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 X 10(-2) X s-1) than for octanoic and linoleic acids (1.5 and 1.2 X 10(-2) X s-1, respectively). Approximately 70, 30, and 25% of the [14C]myristic, [14C]octanoic, or [14C]linoleic acids, respectively, were extracted from the perfusate.