To estimate the functional diameter of alveolar septal microvessels in zone 1, we perfused isolated rat lungs with fluorescent latex particles of specific diameters (0.24, 0.49, 1.05, or 4.0 microns) at pulmonary artery pressures (Ppulmart) that were either 5 or 10 cmH2O less than the air inflation pressure (Pinflat, 25 cmH2O). We then prepared samples for histology. Using a confocal, laser-scanning fluorescence microscope, we measured latex particle densities within the septal plane that ranged from 0.08 +/- 0.04 particles/microns2 (0.24-microns diameter particles) to 0.02 +/- 0.01 particles/microns2 (1.05-microns diameter particles). We found that 4.0-microns diameter particles were not able to enter septa at all. Latex particles were not present in all alveoli When Ppulmart was 5 cmH2O less than Pinflat, 32 +/- 6% of septa contained 0.24-microns diameter particles, but, when Ppulmart was 10 cmH2O less than Pinflat, 5 +/- 6% of septa contained these particles. Percentages were smaller for larger particles. We conclude that, when Ppulmart is both 5 and 10 cmH2O less than Pinflat, the functional diameter of accessible septal microvessels is > 1.05 but < 4.0 microns. Furthermore, the number of accessible septa decreases as the difference between Ppulmart and Pinflat widens.