This study was designed to validate a high-resolution method to measure regional ventilation (V̇a) in small laboratory animals, and to compare regional V̇a and perfusion (Q̇) before and after methacholine-induced bronchoconstriction. A mixture of two different colors of 0.04-μm fluorescent microspheres (FMS) was aerosolized and administered to five anesthetized, mechanically ventilated rats. Those rats also received an intravenous injection of a mixture of two different colors of 15-μm FMS to measure regional blood flow (Q̇). Five additional rats were labeled with aerosol and intravenous FMS, injected with intravenous methacholine, and then relabeled with a second pair of aerosol and intravenous FMS colors. After death, the lungs were reinflated, frozen, and sequentially sliced in 16-μm intervals on an imaging cryomicrotome set to acquire signal for each of the FMS colors. The reconstructed lung images were sampled using randomly placed 3-mm radius spheres. V̇a within each sphere was estimated from the aerosol fluorescence signal, and Q̇ was estimated from the number of 15-μm FMS within each sphere. Method error ranged from 6 to 8% for Q̇ and 0.5 to 4.0% for V̇a. The mean coefficient of variation for Q̇ was 17%, and for V̇a was 34%. The administration of methacholine altered the distribution of both V̇a and Q̇ within lung regions, with a change in V̇a distribution nearly twice as large as that seen for Q̇. The methacholine-induced changes in V̇a were not associated with compensatory shifts in Q̇. Cryomicrotome images of FMS markers provide a high-resolution, anatomically specific means of measuring regional V̇a/Q̇ responses in the rat.