Lymphocyte proliferation and cytokine responses induced by a panel of mycobacterial antigens were compared in Portuguese donors with pulmonary tuberculosis (TB) with or without HIV co-infection, HIV+ patients and healthy Mantoux-positive controls. Control donors showed stronger proliferative responses than any of the patient groups, with secreted antigens (Mycobacterium tuberculosis (Mtb) 30 kD and short-term culture filtrate proteins (ST-CFP)), purified protein derivative (PPD) and Mtb H37Rv Sonicate (MtbS) inducing the strongest proliferation. Patients with pulmonary TB showed lower proliferation to PPD or to the 30-kD antigen. Responses to all the antigens (PPD, ST-CFP, MtbS, 70 kD, 65 kD, 38 kD, 30 kD and 10 kD) were higher in TB/HIV patients with CD4 counts > or = 200 CD4+ T cells/mm3 compared with HIV alone (CD4 > or = 200 T cells/mm3), but were lost in both TB/HIV and HIV patients when CD4 counts fell below 200 T cells/mm3. Measurements of interferon-gamma (IFN-gamma) in culture supernatants revealed that PPD, 30 kD, MtbS and ST-CFP induced the strongest Th1 response. Analysis of mRNA for IFN-gamma, IL-4 and IL-10 confirmed that IFN-gamma production was maintained in patients with pulmonary TB without any concomitant increase in IL-4 or IL-10 mRNA expression, although expression of IL-10 mRNA was increased if HIV infection was present. These results reveal that IFN-gamma production is retained in pulmonary TB patients to a broad range of mycobacterial antigens, and that no switch to IL-4 production is seen even with HIV infection. Secreted antigens, and in particular ST-CFP, were the best inducers of IFN-gamma secretion, confirming their role in protective responses to Mtb.