The nature of brain interstitial fluid (ISF) has long been a subject of controversy. Most of the previous studies on brain ISF were carried out in vitro. In the present study, a novel method was developed to characterize ISF in the living rat brain by magnetic resonance (MR) imaging using gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) as a tracer. Sprague Dawley rats (n=8) were subjected to MR scanning before and after the introduction of Gd-DTPA into the caudate nucleus. A one-way drainage of brain ISF was demonstrated on the dynamic MR images. According to the traditional diffusion model, the diffusion and clearance rate constants of the tracer within brain extracellular space (ECS) were derived as (3.38+-1.07)×10(-4) mm(2) s(-1) and (7.60±4.18)×10(-5) s(-1). Both diffusion and bulk flow contributed to the drainage of ISF from the caudate nucleus, which demonstrated an ISF-cerebrospinal fluid confluence in the subarachnoid space at the lateral and ventral surface of the brain cortex at 3 h after the injection. By using this newly developed method, the brain ECS and ISF can be quantitatively measured simultaneously in the living brain, which will enhance the understanding of ISF and improve the efficiency of drug therapy via the brain interstitium.