This study was designed to validate a high-resolution method to measure regional ventilation (VA) in small laboratory animals, and to compare regional Va and perfusion (Q) before and after methacholine-induced bronchoconstriction. A mixture of two different colors of 0.04-microm 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-microm FMS to measure regional blood flow (Q). 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-microm 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. Va within each sphere was estimated from the aerosol fluorescence signal, and Q was estimated from the number of 15-microm FMS within each sphere. Method error ranged from 6 to 8% for Q and 0.5 to 4.0% for Va. The mean coefficient of variation for Q was 17%, and for Va was 34%. The administration of methacholine altered the distribution of both VA and Q within lung regions, with a change in Va distribution nearly twice as large as that seen for Q. The methacholine-induced changes in Va were not associated with compensatory shifts in Q. Cryomicrotome images of FMS markers provide a high-resolution, anatomically specific means of measuring regional VA/Q responses in the rat.