Time consuming classical diagnostic tests and the increasing incidence of tuberculosis epidemics have rendered the need for new more sensitive diagnostic tools, urgent. This study explored the possibility of a direct and rapid method for the identification and characterization of pathogenic typical and atypical species of mycobacteria from sputum, based on a multiplex PCR assay. Gene bank search on the mycobacterial genome revealed specific sequences that fulfilled the above set criteria. Two pairs of primers were used to amplify a 243 bp fragment of the gene encoding the immunogenic protein MPB 64 and a 133 bp fragment of the gene encoding the 65-KDa mycobacterial antigen. The first pair of primers was selected among others, to detect specifically bacteria of the M. tuberculosis complex, whereas the second, to detect in addition to the latter, those of the M. avium-intracellular complex. Our mutiplex PCR assay, detected and identified correctly, all the mycobacterium tuberculosis and M. avium-intracellulare complex strains provided on pure culture as controls with a sensitivity of 10(-3) colony forming units. Furthermore, by performing our assay on 55 sputum samples from patients with positive culture and Ziehl-Neelsen staining, we identified mycobacterial DNA sequences in all--39 samples with M. tuberculosis complex and 16 with M. avium-intracellulare complex. Out of 300 sputum specimens from patients with clinical evidence of tuberculosis, 149 were positive by our method (95 M. tuberculosis and 54 M. avium-intracellulare complex) whereas 157 samples (95 M. tuberculosis complex, 59 M. avium-intracellulare complex, 1 M. xenopi, and 2 that could not be positively identified) were culture positive and only 95 Ziehl Neelsen positive. These findings suggest that the method described can be applied on sputum, and can identify in one step strains of the M. tuberculosis and M. avium-intracellulare complex and has effectivness comparable to culture methodologies.