Although bulk flow of brain interstitial fluid (ISF) occurs with changes in hydrostatic and osmotic pressures, under normal conditions only diffusion of molecules in the ISF has been reported. Extrachoroidal cerebrospinal fluid (CSF) production and intracerebral injection studies, however, provide indirect evidence for the bulk flow of ISF under normal conditions. We studied tissue penetration profiles of an extracellular molecule in gray and white matter after 1-, 2-, 3-, and 4-h ventriculocisternal perfusions. Gray matter apparent diffusion coefficients were similar at different times as expected with diffusion; however, white matter coefficients decreased significantly with time, suggesting bulk flow of ISF. White matter data was reanalyzed for both bulk flow and diffusion; we calculated a diffusion coefficient of 3.00 x 10(-6) cm2/s and a velocity for ISF of 10.5 micrometers/min toward the ventricle. Additional animals were given 20% mannitol (1.5--3 g/kg) intravenously prior to a /-h ventriculocisternal perfusion. Mannitol produced a significant bulk flow of ISF away from the ventricle in gray matter. We estimate that 30% of extrachoroidal CSF production is from flow of ISF in white matter.