Characterizing lymphocyte subsets in bronchoalveolar lavage fluid (BALF) by flow cytometry (FC) proper gating of the lymphocyte subpopulation being analyzed is crucial. In order to test lymphocyte gate quality for the first time we used a DNA-dye to evaluate plasmamembrane integrity and thus to mark off fluorescent but not DNA-containing particles (eg, debris). A comparative prospective study between this newly developed FC technique and a standard peroxidase anti-peroxidase (PAP) method was performed. Samples of BALF from 50 patients with various pulmonary diseases were examined. After determination of the total cell yield, a differential cell count was performed. Subsequently, the immunophenotype of pan T lymphocyte CD3-, T-helper lymphocyte CD4-, and T-suppressor lymphocyte CD8-positive lymphocyte subsets was assessed with FC as well as with the PAP method. Both methods showed excellent correlation (CD3: r = 0.81; CD4: r = 0.97; CD8: r = 0.96; p < 0.05, respectively). Comparing the mean +/- SEM, FC tends to overestimate CD3+ cells (90.6 +/- 1.0% vs 85.8 +/- 1.3%). For CD4 (45.0 +/- 3.4% vs 44.4 +/- 3.4%) and CD8 (48.1 +/- 3.5% vs 46.7 +/- 3.5%), there was good agreement. In a clinical setting, the reliability of both methods was equivalent, and FC using a DNA-dye to test lymphocyte gate quality offered a rapid and reliable determination of lymphocyte subsets in BAL.