Instillation of glutaraldehyde into the airways of collapsed lungs is a widely used fixation procedure for analysis of pulmonary structure-function relations. In order to better define the effect of this fixation method on the pulmonary microvasculature, a morphometric analysis was made in rat lungs fixed under different conditions. In one group of animals the lungs were allowed to collapse; then the circulation was suddenly interrupted with a sling placed in the coronary sulcus before instillation of the fixative. In a second group the same procedure was carried out with the exception that the lungs were kept inflated at the time of circulatory arrest. In third group the standard technique was used, i.e., the collapsed lungs were instilled before circulatory arrest. The results show that important parameters of alveolar and septal dimensions, and in particular the capillary surfaces and volumes, are alike regardless of the procedure of fixation. However, in lungs fixed by the standard technique a higher capillary hematocrit (approximately 33%) and a decreased harmonic mean thickness of the plasma barrier were found. This artificial hemoconcentration in the microvasculature resulted in a substantial morphometric overestimate of the diffusing capacity of the lung.