A PCR specific for spacer regions 33 and 34 of the direct repeat region of the Mycobacterium tuberculosis complex was developed to complement the biochemical differentiation of M. tuberculosis, Mycobacterium bovis, M. bovis BCG, and Mycobacterium africanum subtypes I and II. In addition, this approach was incorporated into a multiplex PCR that included primers specific for IS6110 and the 65-kDa antigen gene in order to differentiate members of the M. tuberculosis complex from atypical mycobacteria.