An aerosol less than 2-µm in diameter deposits in the alveolar spaces by gravitationally mediated settling, and the deposition density reflects alveolar ventilation. The limitation associated with the use of a fluorescent label to measure regional ventilation is the small quantity of fluorescence in each microsphere. We can obtain a reproducible ventilation signal from 2 cm3 pieces of either dog or pig lung labeled with different FMS aerosols of 1.0-µm diameter. A 3% solution in water of 1.0-µm diameter carboxylated, latex microspheres (FluoSpheres, Molecular Probes, Inc., Eugene, OR) was aspirated into a constant output atomizer, dried, charge neutralized, and delivered to anesthetized pigs or dogs via a mechanical ventilator for either five or ten minutes. For each FMS administration, two different labels were given as a mixture to permit estimation of the error of the method. The mean signal per piece ranged from four to twenty times the background signal of unlabeled lung tissue, depending on administration time, label chosen and species tested. The correlation between simultaneously administered FMS labels ranged between 0.99 and 0.95. This method provides a reproducible means of taking multiple high-resolution measurements of regional ventilation.