An intravenous injection method to measure cerebral vascular extraction fractions of highly diffusible substances in the rat is described. The brain extraction fractions of 3H-labeled water (E[w]) and ethanol (E[e]) were defined as the ratio of either of those tracers to the freely diffusible reference tracer, 14C-butanol, in the brain, divided by the ratio of the tracers available for the extraction during the time between simultaneous intravenous injection of the tracers and decapitation of the rat. E[w] and E[e] were measured in five regions of brain, including brainstem and cerebellum, under P[a]CO2 conditions ranging from 15 to 85 mm Hg. The extraction fractions for both test tracers were shown to vary with P[a]CO2-induced flow changes according to the equation, In(1-E) = - PS/F. When PS/F values calculated from regional measurements of E[w] and E[e] were plotted versus P[a]CO2, least squares regression equations of the plots could be used to compare permeabilities of both tracers at any given P[a]CO2 value. Ratios of the permeabilities of water and ethanol varied regionally but were relatively constant in a given region under different flow states. This intravenous injection method allows for accurate measurement of the extraction fractions of even highly diffusible tracers under varied flow conditions in all brain regions regardless of arterial blood supply.