Investigations on leukocyte populations in the lung have shown that lymphocytes are found in different anatomical compartments. Lymphocytes can be seen to a different extent in the lung interstitium, the epithelium and lamina propria of the bronchi, the bronchoalveolar space, and the marginal lung vascular bed. Previous studies focused on one compartment only, or a mixture of leukocytes from lung homogenates were prepared. This study compared cellular yields from the lung parenchyma, the bronchoalveolar space, and the perfusate of the lung vasculature of healthy male Lewis rats. All compartments were investigated in the same animal, and seven different lymphocyte subsets, monocytes, and natural killer (NK) cells were analyzed using flow cytometry. It was found that the perfusate contained a high proportion of CD4+ lymphocytes compared to the lung interstitium. A very high proportion of CD4+ lymphocytes in the bronchoalveolar lavage (BAL) expressed markers for "memory" T cells. Compared to the blood, the percentage of B and T cells was much lower in the perfusate, whereas the NK cells and monocytes were more frequent. Analysis of leukocyte subsets within all compartments revealed specific, distinguishable cell compositions. Extraction of interstitial lung cells was performed using two different methods. Enzymatic digestion of the lung tissue was compared with a mechanical disruption method. Hardly any differences were observed between the two methods regarding the distribution of lymphocyte subsets, monocytes, and NK cells. These data document the need to study more than one compartment before extrapolating to lymphocytes in the lung in general. Furthermore, changes in numbers of leukocytes and subsets can now be studied in models of lung infections and immune reactions, including the entry from the blood and intrapulmonary migration from one lung compartment to the other.