In this study highly purified culture filtrate proteins obtained from Mycobacterium tuberculosis strains Erdman and H37Rv were tested for their capacity to stimulate immune T cells in vitro, and to immunize mice in vivo. Analysis of the culture filtrate antigen pool revealed a complex mixture of proteins; after separation of this pool into fractions of defined molecular size using an electrophoretic method, it was found that multiple fractions strongly stimulated interferon-gamma (IFN-gamma) secretion by immune CD4 T cells in vitro. In a further series of experiments mice were given multiple immunizations with the culture filtrate protein pool suspended in emulsions of incomplete Freund's adjuvant. Such mice were as resistant as mice given live bacillus Calmette-Guérin (BCG) vaccine to a low dose aerosol challenge infection with M. tuberculosis, but this resistance waned to low levels by 5 months post-vaccination. Furthermore, experiments using the filtrate antigens to boost or augment immunity induced by the BCG vaccination itself were unsuccessful. These data therefore support the hypothesis that the culture filtrate proteins of M. tuberculosis contain multiple antigens that are strongly recognized by T cells acquired during the initial expression of protective immunity to tuberculosis. Conventional immunization with these purified protein antigens can engender a strong degree of protective immunity, but this immunity is apparently not sustained at the same level as that induced by the live vaccine, perhaps suggesting a lack of suitable stimulation of memory immunity.