The fibronectin-binding 30-kDa alpha Ag is a major secretory protein of growing mycobacteria that stimulates in vitro lymphocyte blastogenesis in most healthy purified protein derivative-positive individuals, but only a minority of patients with active tuberculosis. T cell epitopes of the alpha Ag were assessed using blastogenic responses of PBMC from 12 healthy purified protein derivative-positive subjects to a set of synthetic peptides based on the 325-amino acid sequence of the alpha Ag of Mycobacterium bovis BCG. Because epitope-specific precursor cells are infrequent and randomly distributed, we used Poisson analysis to determine positive responses to 10 micrograms/ml of each peptide in 12 replicate culture wells. Seven immunodominant regions of the alpha Ag were identified. Each subjects responded to at least one of the two most dominant epitopes, which correspond to amino acids 131-155 and 233-257 (from N terminus). Peptides of these two epitopes induced production of IFN-gamma by sorted CD4+ T cells. The immunodominant peptides may have use a components of a vaccine and as tools to study the evolution of the immune response to M. tuberculosis. The two most dominant epitopes both occur in regions of the alpha Ag that differ from those of the atypical pathogens M. avium and M. kansasii. In addition, the M. bovis epitope of amino acids 133-155 differs from that of M. tuberculosis by a single amino acid. It may be possible to exploit the sequence differences for development of diagnostic tests with increased specificity.