We have developed an easy and rapid detection and identification system for the diagnosis of mycobacterial diseases. The system is based on selective amplification by PCR of mycobacteria with primers based on the genes coding for 16S rRNA. During PCR, a label (digoxigenin-11-dUTP) is incorporated with biotinylated species-specific oligonucleotides (oligonucleotide-specific capture plate hybridization [OSCPH]. One oligonucleotide specific for the genus Mycobacterium and seven species-specific (Mycobacterium tuberculosis, M. avium, M. intracellulare, M. scrofulaceum, M. xenopi, M. genavense, and M. chelonae) oligonucleotides were designed as capturing probes. After specific hybridization, an enzyme immunoassay reveals the specifically bound complexes and thus permits identification of the mycobacterium. A total of 70 mycobacterial strains were tested. For 69 strains, results concordant with conventional identification were obtained. One M. chelonae strain was negative with the M. chelonae probe and was later reidentified as M. fortuitum. Moreover, for 15 clinical samples suspected of harboring nontuberculous mycobacteria, OSCPH was able to confirm all culture results and could identify one M. genavense infection for which standard culture results were negative. PCR-OSCPH is easily applicable and much faster than culture. It could become a valuable alternative approach for the diagnosis of mycobacterial infections.