METHODS Alveolar macrophages (AM), a heterogeneous cell population, play a critical role in eliminating mycobacterial infections in collaboration with lymphocytes. Patients with diabetes mellitus (DM) show increased susceptibility to pulmonary tuberculosis (TB) infection. It is still uncertain whether there is a defect in T cell or AM activation in patients with DM against TB infection. OBJECTIVE To study the difference in activation status of AM and T cells between patients with TB + DM and TB alone. METHODS The heterogeneity of AM from 14 patients with TB + DM, 9 with TB alone, 10 normal subjects and 8 DM alone patients, was studied using Percoll density fractionation. The intracellular H2O2 production of AM before and after stimulation with phorbol myristate acetate (PMA) or F-Met-Leu-Phen (FMLP) was assayed by loading cells with 2',7'-dichlorofluorescin (DCFH) and analyzed by flow cytometry. Lymphocytes subsets (CD3, CD4, CD8) and their activation status (CD25) in bronchoalveolar lavage were also measured. RESULTS The proportion of the least dense AM (< 1,030 g/ml) and the magnitude of DCFH oxidation of AM was higher in TB patients than in normal subjects, regardless of DM. Patients with TB + DM had a significantly lower proportion of the least density AM fraction than TB alone patients, regardless of disease extent. Among TB patients, the proportion of the least dense AM was inversely correlated with the bacterial load on sputum and the disease extent on chest radiograph. Stimulation of AM with PMA or FMLP induced an increase in the hypodense AM subpopulations and enhanced intracellular H2O2 generation in patients with TB + DM and to a similar extent in normal subjects, but not in patients with TB alone. There was no significant difference in CD3 numbers, CD4/CD8 ratio, and CD25+ cells between patients with TB alone and TB + DM. The activation status of AM or T lymphocytes from DM alone patients was not significantly different from those from normal subjects. CONCLUSIONS Hypodense subpopulations of AM increase in active TB patients and are related to the disease severity as well as activation status of AM. AM in TB patients complicated with DM was less activated, and may be contributory to the susceptibility to mycobacterial infection.