FGF21 recently has been proposed as a missing link in the biology of fasting, raising the question of whether it directly reaches the brain. We used multiple time-regression analysis to quantify the influx rate of this polypeptide across the blood-brain barrier (BBB), size-exclusion chromatography to examine degradation, capillary depletion to differentiate entry into brain parenchyma from retention in the microvasculature, and measurement of efflux rate to determine a possible confounding effect on measurement of entry. FGF21 was 94% intact in serum and 75% in brain 10 min after intravenous bolus delivery. Its influx rate was 0.23+/-0.12 microl/g-min, nearly four times faster than that of the vascular marker albumin. At 10 min, about 0.5% of the administered FGF21 was present in a gram of brain tissue. Of this, 70% reached the parenchyma of the brain. Co-injection of excess FGF21 failed to inhibit the influx, showing a lack of saturation. Efflux, which occurred at the same rate as the bulk reabsorption of cerebrospinal fluid, also was not saturable. In summary, FGF21 shows significant, non-saturable, unidirectional influx across the BBB.