A tricolor flow cytometric application is described which permits the determination of total T lymphocytes, T helper lymphocytes and cytotoxic T lymphocytes, natural killer cells and activated T lymphocytes under the same experimental conditions. Even when the lymphocyte count is low, when there is contamination by dust particles or when the cells are damaged the method works with high specificity and reliability. Lymphocytes are identified on the basis of their expression of the pan-leucocyte marker CD45, their side scatter, and plasma membrane integrity, assessed using the fluorescent DNA dye LDS 751. When lymphocyte subsets assessed by flow cytometry were compared with the standard immunoperoxidase method, a strong correlation was found for the CD3+, CD4+ and CD8+ cells. A weak correlation was found for CD25+ cells (r = 0.5). No correlation was seen for CD56+ cells. The high specificity of the procedure suggests that it could be used routinely for the analysis of lymphocytes in bronchoalveolar lavage fluid (BALF), especially when the BALF is contaminated by inorganic particles. Furthermore the application may contribute to the evaluation of lymphocyte subset analysis in the presence of low cell counts.